Patent Publication Number: US-2010112551-A1

Title: Approaches to identifying mutations associated with hereditary nonpolyposis colorectal cancer

Description:
FIELD OF THE INVENTION 
     The present invention relates to the field of genetic screening and diagnostics. More specifically, the described embodiments concern methods to screen multiple samples, in a single assay, for the presence or absence of mutations or polymorphisms that relate to Hereditary Nonpolyposis Colorectal Cancer (HNPCC). 
     BACKGROUND OF THE INVENTION 
     Hereditary Nonpolyposis Colorectal Cancer (HNPCC) is the most common hereditary form of colon cancer. It is a genetic syndrome caused by mutations in any one of five or more genes that code for proteins involved with repair of damaged or aberrant DNA, two of which are the human mismatch repair genes mutL homolog 1 (MLH1) and mutS homologue 2 (MSH2). Individuals that inherit mutations associated with HNPCC are at a much higher risk for colon cancer than the general population (80% chance of developing color cancer, vs. 4%) and at an earlier age (average age of onset of colon cancer: 44 years old, vs. 65 years of age for the general population). Individuals with HNPCC also have a higher risk of getting certain other forms of cancer (Lynch, H. et al. Cancer 78:1149 (1996)). There is a great need for approaches to identify mutations and polymorphisms that relate to this deadly disease. 
     Current DNA-based diagnostics allow for the identification of a single mutation or polymorphism or gene per analysis. Although high-throughput methods and gene chip technology have enabled the ability to screen multiple samples or multiple loci within the same sample, these approaches require several independent reactions, which increases the time required to process clinical samples and drastically increases the cost. Further, because of time and expense, conventional diagnostic approaches focus on the identification of the presence of DNA fragments that are associated with a high frequency of mutation, leaving out analysis of other loci that may be critical to diagnose a disease. The need for more approaches for the diagnosis of genetic disease is manifest. 
     With the advent of multiplex Polymerase Chain Reaction (PCR), the ability to use multiple primer sets to generate multiple extension products from a single gene is at hand. By hybridizing isolated DNA with multiple sets of primers that flank loci of interest on a single gene, it is possible to generate a plurality of extension products in a single PCR reaction corresponding to fragments of the gene. As the number of primers increases, however, the complexity of the reaction increases and the ability to resolve the extension products using conventional techniques fails. Further, since many diseases are caused by changes of a single nucleotide, the rapid detection of the presence or absence of these mutations or polymorphisms is frustrated by the fact that the PCR products that indicate both the diseased and non-diseased state are of the same size. 
     Developments in gel electrophoresis and high performance liquid chromatography (HPLC), however, have enabled the separation of double-stranded DNAs based upon differences in their melting behaviors, which has allowed investigators to resolve DNA fragments having a single mutation or single polymorphism. Techniques such as temporal temperature gradient gel electrophoresis (TTGE) and denaturing high performance liquid chromatography (DHPLC) have been used to screen for small changes or point mutations in DNA fragments. 
     The separation principle of TTGE, for example, is based on the melting behavior of DNA molecules. In a denaturing polyacrylamide gel, double-stranded DNA is subject to conditions that will cause it to melt in discrete segments called “melting domains.” The melting temperature Tm of these domains is sequence-specific. When the Tm of the lowest melting domain is reached, the DNA will become partially melted, creating branched molecules. Partial melting of the DNA reduces its mobility in a polyacrylamide gel. Since the Tm of a particular melting domain is sequence-specific, the presence of a mutation or polymorphism will alter the melting profile of that DNA in comparison to the wild-type or non-polymorphic DNA. That is, a heteroduplex DNA consisting of a wild-type or non-polymorphic strand annealed to mutant or poymorphic strand, will melt at a lower temperature than a homoduplex DNA strand consisting of two wild-type or non-polymorphic strands. Accordingly, the DNA containing the mutation or polymorphism will have a different mobility compared to the wild-type or non-polymorphic DNA. 
     Similarly, the separation principle of DHPLC is based on the melting or denaturing behavior of DNA molecules. As the use and understanding of HPLC developed, it became apparent that when HPLC analyses were carried out at a partially denaturing temperature, i.e., a temperature sufficient to denature a heteroduplex at the site of base pair mismatch, homoduplexes could be separated from heteroduplexes having the same base pair length. (See e.g., Hayward-Lester, et al., Genome Research 5:494 (1995); Underhill, et al., Proc. Natl. Acad. Sci. USA 93:193 (1996); Oefner, et al., DHPLC Workshop, Stanford University, Palo Alto, Calif., (Mar. 17, 1997); Underhill, et al., Genome Research 7:996 (1997); Liu, et al., Nucleic Acid Res., 26:1396 (1998), all of which and the references contained therein are hereby expressly incorporated by reference in their entireties). 
     Techniques such as Matched Ion Polynucleotide Chromatography (MIPC) and Denaturing Matched Ion Polynucleotide Chromatography (DMIPC) have also been employed to increase the sensitivity of detection. It was soon realized that DHPLC, which for the purposes of this disclosure includes but is not limited to, MIPC, DMIPC, and ion-pair reverse phase high-performance liquid chromatography, could be used to separate heteroduplexes from homoduplexes that differed by as little as one base pair. Various DHPLC techniques have been described in U.S. Pat. Nos. 5,795,976; 5,585,236; 6,024,878; 6,210,885; Huber, et al., Chromatographia 37:653 (1993); Huber, et al., Anal. Biochem. 212:351 (1993); Huber, et al., Anal. Chem. 67:578 (1995); ODonovan et al., Genomics 52:44 (1998), Am J Hum Genet. December; 67(6):1428-36 (2000); Ann Hum Genet. September:63 (Pt 5):383-91 (1999); Biotechniques, April; 28(4):740-5 (2000); Biotechniques. November; 29(5):1084-90, 1092 (2000); Clin Chem. August; 45(8 Pt 1):1133-40 (1999); Clin Chem. April; 47(4):635-44 (2001); Genomics. August 15; 52(1):44-9 (1998); Genomics. March 15; 56(3):247-53 (1999); Genet Test.; 1(4):237-42 (1997-98); Genet Test.:4(2):125-9 (2000); Hum Genet. June; 106(6):663-8 (2000); Hum Genet. November; 107(5):483-7 (2000); Hum Genet. November; 107(5):488-93 (2000); Hum Mutat. December; 16(6):518-26 (2000); Hum Mutat. 15(6):556-64 (2000); Hum Mutat. March; 17(3):210-9 (2001); J Biochem Biophys Methods. November 20; 46(1-2):83-93 (2000); J Biodhem Biophys Methods. January 30; 47(1-2):5-19 (2001); Mutat Res. November 29; 430(1):13-21 (1999); Nucleic Acids Res. March 1; 28(5):E13 (2000); and Nucleic Acids Res. October 15; 28(20):E89 (2000), all of which, including the references contained therein, are hereby expressly incorporated by reference in their entireties. Despite the efforts of many, there remains a need for more approaches to screen and identify mutations and/or polymorphisms in genes, in particular, genes that relate to Hereditary Nonpolyposis Colorectal Cancer. 
     SUMMARY OF THE INVENTION 
     Aspects of the invention concern rapid and inexpensive but efficient approaches to determine the presence or absence of mutations and/or polymorphisms that relate to Hereditary Nonpolyposis Colorectal Cancer (HNPCC). Several oligonucleotide primers specific for the human mismatch repair genes, mutL homolog 1 (MLH1) and mutS homologue 2 (MSH2), have been developed (e.g., Tables A and 2). These primers and oligonucleotides that are any number between 1-75 nucleotides upstream or downstream of said primers are unique in sequence and in their ability to generate extension products that melt evenly over vast stretches of nucleotides, which greatly improves the sensitivity of detection (e.g., single base mutations). It was then realized that by grouping extension products with similar melting behaviors, one can rapidly and efficiently separate multiple extension products on the basis of melting behavior on the same lane of a TTGE gel or in the same run on a DHPLC. Accordingly, a rapid, inexpensive and efficient approach to diagnose a subject at risk for HNPCC was discovered, whereby extension products are generated from a subject&#39;s DNA using the primers described herein, the extension products are grouped or mixed according to their melting behavior, and the grouped or mixed extension products are separated on the basis of melting behavior (e.g., one group per lane of TTGE gel). Not only does the pooling of extension products reduce cost and the time to perform the analysis but, because the extension products are optimized for melting behavior, the sensitivity of detection remains very high. 
     By one approach, for example, a method of identifying the presence or absence of a genetic marker in the human mismatch repair genes MLH1 and MSH2 of a subject is conducted by providing a DNA sample from said subject; providing at least one primer set from Table A; contacting said DNA and said at least one primer set; generating an extension product from said at least one primer set that comprises a region of DNA that includes the location of said genetic marker; separating said extension product on the basis of melting behavior; and identifying the presence or absence of said genetic marker in said subject by analyzing the melting behavior of said extension product. In related embodiments, at least 2, 3, 4, 5, 6, 7, or 8 primer sets from Table A are contacted with said DNA. In more related embodiments, the extension products generated from said 2, 3, 4, 5, 6, 7, or 8 primer sets are grouped according to Table D and separated on the basis of melting behavior. Optionally, the extension products and/or the sample nucleic acid used in the approaches above can be sequenced so as to verify and/or identify the mutation or polymorphism. 
     In another set of embodiments, a method of identifying the presence or absence of a genetic marker in the human mismatch repair genes mutL homolog 1 (MLH1) and mutS homologue 2 (MSH2) of a subject is conducted by providing a DNA sample from said subject; providing at least one primer set that is any number between 1-75 nucleotides upstream or downstream of a primer set from Table A; contacting said DNA and said at least one primer set; generating an extension product from said at least one primer set that comprises a region of DNA that includes the location of said genetic marker, separating said extension product on the basis of melting behavior; and identifying the presence or absence of said genetic marker in said subject by analyzing the melting behavior of said extension product. In related embodiments, at least 2, 3, 4, 5, 6, 7, or 8 primer sets from Table A are contacted with said DNA. In more related embodiments, the extension products generated from said 2, 3, 4, 5, 6, 7, or 8 primer sets are grouped according to Table D and separated on the basis of melting behavior. As above, optionally, the extension products and/or the sample nucleic acid used in these approaches can be sequenced so as to verify and/or identify the mutation or polymorphism. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a melting curve for the extension product MLH1 2A spanning the beginning of exon 2 and nucleotides ˜100-188 of the depicted fragment. The x axis shows the number of nucleotides and the y axis shows the temperature. 
         FIG. 2  shows a melting curve for the extension product MLH1 2B covering the end of exon 2 and nucleotides ˜100-171 of the depicted fragment. The x axis shows the number of nucleotides and the y axis shows the temperature. 
         FIG. 3  shows a melting curve for the extension product MSH2 9 covering exon 9 and nucleotides ˜100-260 of the depicted fragment. The x axis shows the number of nucleotides and the y axis shows the temperature. 
         FIG. 4  shows a melting curve for the extension product MSH2 15 covering exon 15 and nucleotides ˜48-230 of the depicted fragment. The x axis shows the number of nucleotides and the y axis shows the temperature. 
         FIG. 5  shows a melting curve for the extension product MLH1 3A spanning the beginning of exon 3 and nucleotides ˜100-218 of the depicted fragment. The x axis shows the number of nucleotides and the y axis shows the temperature. 
         FIG. 6  shows a melting curve for the extension product MLH1 3B spanning the end of exon 3 and nucleotides ˜23-130 of the depicted fragment. The x axis shows the number of nucleotides and they axis shows the temperature. 
         FIG. 7  shows results from experiments using primers with fluorescent tags to amplify portions of exon 10 of the Cystic Fibrosis Transmembrane Regulator (CTFR) gene. Two polymorphisms were amplified in this experiment: deltaF508 (DF508) and M470V. These results reveal the homozygous state of the clinical DNA samples used in the reactions when the products are mixed with wildtype DNA before analysis via TTGE. Texas Red (tr) and Oregon Green (og) tags are used. Banding patterns for wild type (WT), heterozygous (HET), homozygous (HOMO) and mixtures of these patterns (in the right hand side lanes, containing mixtures of tr and og products) are displayed. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Embodiments described herein concern a novel approach to screen for the presence or absence of multiple mutations or polymorphisms in a plurality of genes, in particular, genes associated with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). Particularly preferred embodiments concern approaches to screen multiple loci in the human mismatch repair genes mutL homolog 1 (MLH1) and mutS homologue 2 (MSH2) so as to determine the presence or absence of a mutation or polymorphism that may indicate a suseptibility to Hereditary Nonpolyposis Colorectal Cancer (HNPCC) and/or other cancers. Similar approaches have been used to identify the presence or absence or polymorphisms or mutations related to cystic fibrosis, which are described in U.S. patent application Ser. Nos. 10/300,683; 60/333,351; and 60/486,864, all of which are hereby expressly incorporated by reference in their entireties. 
     Several embodiments permit very sensitive detection of single base mutations, single base mismatches, and small nuclear polymorphisms (SNPs), as well as, larger alterations in DNA at multiple loci, in a plurality of genes, in multiple samples. Additionally, by employing a DNA standard or by screening a plurality of DNA samples in the same assay, improved sensitivity of detection can be obtained. A novel approach to designing primers and extension products generated therefrom is described in the context of an assay that was performed to detect the presence or absence of genetic markers, polymorphisms, or mutations on the human mismatch repair genes mutL homolog 1 (MLH1) and mutS homologue 2 (MSH2). By identifying the presence or absence of these polymorphisms or mutations, an understanding of susceptibility to Hereditary Nonpolyposis Colorectal Cancer (HNPCC) can be obtained. 
     Embodiments include methods of identifying the presence or absence of a plurality of genetic markers in a subject in the same gene or separate genes. One method is practiced, for example, by providing a DNA sample from said subject, providing a plurality of nucleic acid primer sets that hybridize to said DNA at regions that flank said plurality of genetic markers, wherein each primer set has a first and a second primer and, wherein said plurality of genetic markers exist on the same gene or a plurality of genes, contacting said DNA and said plurality of nucleic acid primer sets in a single reaction vessel or multiple reaction vessels, generating, in said reaction vessel(s), a plurality of extension products that comprise regions of DNA that include the location of said plurality of genetic markers, separating said plurality of extension products on the basis of melting behavior in a single lane or multiple lanes of a gel or a single run or multiple runs on a column, and identifying the presence or absence of said plurality of genetic markers in said subject by analyzing the melting behavior of said plurality of extension products. In some aspects of this method the separation on the basis of melting behavior is accomplished by TTGE and in other embodiments the separation on the basis of melting behavior is accomplished by DHPLC. In some embodiments, said extension products are first separated by size for a period sufficient to separate populations of extension products and then separated by melting behavior. The size separation can be accomplished on the TTGE gel or DHPLC column prior to separating on the basis of melting behavior. 
     Preferably, after generating the extension products by an amplification technique (e.g., Polymerase Chain Reaction or PCR), the extension products are grouped and pooled according to their predicted and/or actual melting behavior. In this way, multiple extension products, which correspond to different regions on the same gene or different&#39; regions on a plurality of genes can be separated on the same lane of a TTGE gel or in the same run on a DHPLC column. By carefully designing the primers, such that the extension products generated therefrom melt over large stretches of DNA (approximately 25, 50, 75, 100, 125, or 150 nucleotides) at roughly the same temperature (within up to 1.5° C. of one another), it was unexpectedly discovered that multiple extension products (2, 3, 4, 5, 6 or more) can be separated on the same lane of a TTGE gel or in the same run on an DHPLC column, thereby substantially reducing the cost of conducting the analysis and increasing the speed of analysis. 
     In some embodiments, either the first or the second primer comprise a GC clamp. In other aspects of this embodiment, either the first or the second primer hybridize to a sequence within an intron. Preferably, at least one of the plurality of genetic markers is indicative of Hereditary Nonpolyposis Colorectal Cancer (HNPCC). In other embodiments, the plurality of primer sets consist of at least 3, 4, 5, 6, or 7 primer sets. Additionally, in some embodiments, the plurality of genes consist of at least 2, 3, 4, 5, 6, or 7 genes that are related to Hereditary Nonpolyposis Colorectal Cancer (HNPCC). The method above preferably generates the extension products using the Polymerase Chain Reaction (PCR) and the method can be supplemented by a step in which a control DNA is added. 
     Another embodiment concerns a method of identifying the presence or absence of a plurality of genetic markers in a plurality of subjects. This method is practiced by: providing a DNA sample from said plurality of subjects, providing a plurality of nucleic acid primer sets that hybridize to said DNA at regions that flank said plurality of genetic markers, wherein each primer set has a first and a second primer and, wherein said plurality of genetic markers exist on the same gene or on a plurality of genes, contacting said DNA and said plurality of nucleic acid primer sets in a single reaction vessel or multiple vessels, generating, in said reaction vessel(s), a plurality of extension products that comprise regions of DNA that include the location of said plurality of genetic markers, separating said plurality of extension products on the basis of melting behavior in a single lane or multiple lanes of a gel or a single run or multiple runs on a column, and identifying the presence or absence of said plurality of genetic markers in said plurality of subjects by analyzing the melting behavior of said plurality of extension products. In some aspects of this embodiment, the separation on the basis of melting behavior is accomplished by TTGE and in other embodiments the separation on the basis of melting behavior is accomplished by DHPLC. Again, preferred genetic markers for identification using the approaches above, concern genes that are associated with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). 
     As above, preferably, after generating the extension products by the amplification technique (e.g., PCR) from the plurality of subjects, the extension products are grouped and pooled according to their predicted and/or actual melting behavior. By separating multiple extension products generated from a plurality of subjects in the same lane of a TTGE gel or in the same run on a DHPLC column, the cost of analysis is substantially reduced. Because the incidence of polymorphism or mutation in the population as a whole is small, the large scale screening, described above, can be performed. When a polymorphism and/or mutation is detected in this type of assay, single subject assays can be performed, as described above, to identify the subject(s) that have the polymorphism and/or mutation. Optionally, the extension products and/or the nucleic acid samples themselves can be sequenced so as to verify and/or identify the mutation or polymorphism. 
     In more embodiments, the plurality of subjects consist of at least 2, 3, 4, 5, 6, or 7 subjects. In more aspects of this embodiment, the plurality of primer sets consist of at least 3, 4, 5, 6, or 7 primer sets. Additionally, in some embodiments, the plurality of genes consist of at least 2, 3, 4, 5, 6, or 7 genes. The method above preferably generates the extension products using PCR and the method can be supplemented by a step in which a control DNA is added. 
     Still another embodiment involves a method of identifying the presence or absence of a mutation or polymorphism in a subject related to Hereditary Nonpolyposis Colorectal Cancer (HNPCC). This method is practiced by: providing a DNA sample from said subject, generating a population of extension products from said sample, wherein said extension products comprise a region of said DNA that corresponds to the location of said mutation or polymorphism, providing at least one control DNA, wherein said control DNA corresponds to the extension product but lacks said mutation or polymorphism, contacting said control DNA and said population of extension products in a single reaction vessel, thereby forming a mixed DNA sample, heating said mixed DNA sample to a temperature sufficient to denature said control DNA and said DNA sample, cooling said mixed DNA sample to a temperature sufficient to anneal said control DNA and said DNA sample, separating said mixed sample on the basis of melting behavior in a single lane or multiple lanes of a gel or a single run or multiple runs on a column, and identifying the presence or absence of said mutation or polymorphism by analyzing the melting behavior of said mixed DNA sample. 
     By this approach, the addition of the control DNA followed by the heating and cooling steps, forces heteroduplex formation, if a polymorphism or mutation is present, which facilitates identification. In some aspects of this embodiment, the control DNA is DNA obtained or amplified from a second subject and the presence or absence of a mutation or polymorphism is known. In other aspects of the invention, heteroduplex formation can be forced by pooling the extension products generated from a plurality of subjects and denaturing and annealing, as above. Because the predominant genotype in a plurality of subjects lacks polymorphisms or mutations in the gene(s) analyzed, the majority of the DNA will force heteroduplex formation with any polymorphic or mutant DNA in the pool. Accordingly, the identification of mutant and/or polymorphic DNA is facilitated and the cost of the analysis is reduced. In some aspects of this embodiment, the separation on the basis of melting behavior is accomplished by TTGE and in other embodiments the separation on the basis of melting behavior is accomplished by DHPLC. 
     Still more embodiments concern the primers or groups of primers disclosed herein (preferably MLH1 and MSH2 specific primers), extension products generated from said primers, kits containing said nucleic acids, and methods of using these primers, groups of primers, or extension products to diagnose a risk for a disease (e.g., HNPCC). These nucleic acid primers can be used to efficiently determine the presence or absence of a polymorphism or mutation in a multiplex PCR reaction that screens a plurality of genes and a plurality of subjects in a single reaction vessel or multiple reaction vessels. Additionally, reaction vessels comprising a DNA sample, and a plurality of nucleic acid primer sets that hybridize to said DNA sample at regions that flank a plurality of genetic markers, wherein said plurality of genetic markers exist on a single gene or a plurality of genes are embodiments. Further, a reaction vessel comprising a plurality of DNA samples obtained from a plurality of subjects and a plurality of nucleic acid primer sets that hybridize to said plurality of DNA samples at regions that flank a plurality of genetic markers, wherein said plurality of genetic markers exist on a plurality of genes or on a single gene are embodiments. 
     Still more aspects of the invention include a reaction vessel containing a plurality of extension products (2, 3, 4, 5, 6, 7, 8, 9, or 10 or more), which melt at approximately the same temperature (e.g., 0° C.-1.5° C. from one another). That is, in some approaches, the extension products are generated in separate vessels using individual primers sets but the extension products with similar melting behaviors are pooled prior to loading onto a TTGE gel or DHPLC. The pooled extension products are loaded onto a single lane of a gel and resolved by melting behavior. In some embodiments, differing fluorescent labels are employed in the individual PCR reactions so that the extension products generated therefrom fluoresce at different wavelengths (e.g., produce a different color under a detector) so as to facilitate identification after the pooled extension products are resolved on the gel or column. 
     Other embodiments concern a gel having lanes and adapted to separate different DNAs comprising a plurality of extension products, in a single lane of said gel, wherein said plurality of extension products melt at approximately the same temperature but are resolvable on said gel and, which correspond to regions of DNA located on a plurality of genes or on a single gene and, wherein said regions of DNA comprise loci that indicate a genetic trait and a gel having lanes and adapted to separate different DNAs comprising a plurality of extension products, in a single lane of said gel, wherein said plurality of extension products correspond to regions of DNA located on a plurality of genes or on a single gene in a single individual or a plurality of subjects and, wherein said regions of DNA comprise loci that indicate a genetic trait. 
     Additional embodiments include a DHPLC column adapted to separate different DNAs comprising a plurality of extension products, wherein said plurality of extension products melt at approximately the same temperature but are resolvable on said column and, which correspond to regions of DNA located on a plurality of genes or a single gene or and, wherein said regions of DNA comprise loci that indicate a genetic trait and a DHPLC column adapted to separate different DNAs comprising a plurality of extension products, wherein said plurality of extension products correspond to regions of DNA located on a plurality of genes or on a single gene in a single individual or a plurality of subjects and, wherein said regions of DNA comprise loci that indicate a genetic trait. More description of the compositions and methods described above is provided in the in the following sections. 
     Approaches to Facilitate and Reduce the Cost of Genetic Analysis 
     Aspects of the invention described herein concern approaches to analyze DNA, samples for the presence or absence of a plurality of genetic markers that reside on a plurality of genes in a single assay. Some embodiments allow one to rapidly distinguish a plurality of DNA fragments in a single sample that differ only slightly in size and/or composition (e.g., a single base change, mutation, or polymorphism). Other embodiments concern methods to screen multiple genes from a subject, in a single assay, for the presence or absence of a mutation or polymorphism. An approach to achieve greater sensitivity of detection of mutations or polymorphisms present in a DNA sample is also provided. Preferred embodiments, however, include methods to screen multiple genes, in a plurality of DNA samples, in a single assay, for the presence or absence of mutations or polymorphisms. 
     It was discovered that multiple extension products that have slight differences in length and/or composition can be resolved by separating the DNA on the basis of melting temperature. By one approach, a plurality of varying lengths of double-stranded DNA are applied to a denaturing gel and the double-stranded DNAs are separated by applying an electrical current while the temperature of the gel is raised gradually. By slowly increasing the temperature while the DNA is electrically separated on a polyacrylamide gel containing a denaturant (e.g., urea), the dsDNA eventually denatures to partially single stranded (branched molecules) DNA. Because branched or heteroduplex DNA migrates more rapidly or more slowly than dsDNA or homoduplex DNA, one can quickly determine the differences in melting behavior between DNA fragments, compare this melting temperature to a standard DNA (e.g., a wild-type DNA or non-polymorphic DNA), and identify the presence or absence of a mutation or polymorphism in the screened DNA. This technique efficiently separates multiple DNA fragments, generated by a single multiplex PCR reaction on a plurality of loci from different genes (e.g., in one experiment, 10 different loci were analyzed in the same reaction and each of the extension products, some that differed by only a single mutation, were efficiently resolved). 
     It was also discovered that multiple extension products that have slight differences in length and/or composition can be resolved by separating the DNA by DHPLC. By one approach, a plurality of varying lengths of double-stranded DNA are applied to a ion-pair reverse phase HPLC column (e.g., alkylated non-porous poly(styrene-divinylbenzene)) that has been equilibrated to an appropriate denaturing temperature, depending on the size and composition of the DNA to be separated (e.g., 53° C. to 63° C.) in an appropriate buffer (e.g., 0.1 mM triethylamine acetate (TEAA) pH 7.0). Once applied to the column, the double stranded DNA binds to the matrix. By slowly increasing the presence of a denaturant (e.g., acetonitrile in TEAA), the dsDNA eventually denatures to partially single stranded (branched molecules) DNA and elutes from the column. Preferably a linear gradient is used to slowly elute the bound DNA. Detection can be accomplished using a U.V. detector, radioactivity, dyes, or fluorescence. In some embodiments, the extension products are first separated on the basis of size using a shallow gradient of denaturant for a time sufficient to separate individual populations of extension products and then on the basis of melting behavior using a deeper gradient of denaturant. The techniques described in the following references can also be modified for use with aspects of the invention: U.S. Pat. Nos. 5,795,976; 5,585,236; 6,024,878; 6,210,885; Huber, et al., Chromatographia 37:653 (1993); Huber, et al., Anal. Biochem. 212:351 (1993); Huber, et al., Anal. Chem. 67:578 (1995); ODonovan et al., Genomics 52:44 (1998), Am J Hum Genet. December; 67(6):1428-36 (2000); Ann Hum Genet. September:63 (Pt 5):383-91 (1999); Biotechniques, April; 28(4):740-5 (2000); Biotechniques. November; 29(5):1084-90, 1092 (2000); Clin Chem. August; 45(8 Pt 1):1133-40 (1999); Clin Chem. April; 47(4):635-44 (2001); Genomics. August 15; 52(1):44-9 (1998); Genomics. March 15; 56(3):247-53 (1999); Genet Test.; 1(4):237-42 (1997-98); Genet Test:4(2):125-9 (2000); Hum Genet. June; 106(6):663-8 (2000); Hum Genet. November; 107(5):483-7 (2000); Hum Genet. November; 107(5):488-93 (2000); Hum Mutat. December; 16(6):518-26 (2000); Hum Mutat. 15(6):556-64 (2000); Hum Mutat. March; 17(3):210-9 (2001); J Biochem Biophys Methods. November 20; 46(1-2):83-93 (2000); J Biochem Biophys Methods. January 30; 47(1-2):5-19 (2001); Mutat Res. November 29; 430(1):13-21 (1999); Nucleic Acids Res. March 1; 28(5):E13 (2000); and Nucleic Acids Res. October 15; 28(20):E89 (2000), all of which are hereby expressly incorpo&#39;rated by reference in their entireties including the references cited therein. 
     Because branched or heteroduplex DNA elutes either more rapidly or more slowly than homoduplex DNA, one can quickly determine the differences in melting behavior between DNA fragments, compare this melting temperature to a standard DNA (e.g., a wild-type or non-polymorphic homoduplex DNA), and identify the presence or absence of a mutation or polymorphism in the screened DNA. This technique efficiently separates multiple DNA fragments, generated by a single multiplex PCR reaction on a plurality of loci from different genes. 
     Some of the embodiments described herein have adapted the DNA separation techniques described above to allow for high-throughput genetic screening of organisms (e.g., plant, virus, bacteria, mold, yeast, and animals including humans). Typically, multiple primers that flank genetic markers (e.g., mutations or polymorphisms that indicate a congenital disease or a trait) on different genes are employed in a single amplification reaction or multiple amplification reactions and the multiple extension products are separated on a denaturing gel or by DHPLC according to their melting behavior. The presence or absence of mutations or polymorphisms, also referred to as “genetic markers”, in the subject&#39;s DNA are then detected by identifying an aberrant melting behavior in the extension products (e.g., migration on a gel that is too fast or too slow or elution from a DHPLC column that is too fast or too slow). Advantageously, some embodiments provide a greater understanding of a subject&#39;s health because more loci that are indicative of disease, for example, are analyzed in a single assay. Further, some embodiments drastically reduce the cost of performing such diagnostic assays because many different genes and markers for disease can be screened simultaneously in a single assay. 
     By one approach, for example, a biological sample from the subject (e.g., blood) is obtained by conventional means and the DNA is isolated. Next, the DNA is hybridized with a plurality of nucleic acid primers that flank regions of a plurality of genetic loci or markers that are associated with or linked to the plurality of traits to be analyzed. Although 10 different loci have been detected in a single assay (requiring 20 primers), more or less loci can be screened in a single assay depending on the needs of the user. Preferably, each assay has sufficient primers to screen at least three different loci, which may be located on three different genes. That is, the embodied assays can employ sufficient primers to screen at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24 or more, independent loci or markers that are indicative of a disease in a single assay (e.g., in the same tube or multiple tubes) and these loci can be on different genes. Because more than one loci or marker can be detected by a single set of primers, the detection of 20 different markers, for example, can be accomplished with less than 40 primers. However, in many assays, a different set of primers is needed to detect each different loci. Thus, in several embodiments, at least 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, or more primers are used. 
     Desirably, the primers hybridize to regions of human DNA that flank markers or loci associated with or linked to human diseases such as: familial hypercholesterolemia (FH), cystic fibrosis, Tay-sachs, thalassemia, sickle cell disease, phenylketonuria, galactosemia, fragile X syndrome, hemophilia A, myotonic dystrophy, medium-chain acyl CoA dehydrogenase, maturity onset diabetes, cystinuria, methylmolonic acidemia, urea cycle disorders, hereditary fructose intolerance, hereditary hemachromatosis, neonatal thrombocytopenia, Gaucher&#39;s disease, tyrosinemia, Wilson&#39;s disease, alcaptonuria, hypolactasia, Baker&#39;s disease, argininemia Adenomatous polyposis coli (APC), Adult Polycystic Kidney disease, a-1-antitrypsin deficiency, Duchenne Muscular Dystrophy, Hemophilia A, Hereditary Nonpolyposis colorectal cancer, Huntingtons disease, Marfan syndrome, Myotonic dystrophy, Neurofibromatosis, Osteogenesis imperfecta, Retinoblastoma, Sickle cell disease, Freidrichs ataxia, Hemoglobinopathies, Leber&#39;s hereditary optic neuropathy, MCAD, Canavan&#39;s disease, Retintitus Pigmentosa, Bloom Syndrome, Fanconi anemia, and Neimann Pick disease. It is particularly preferred that the primers hybridize to regions of DNA that flank markers associated with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). It should be understood, however, that the list above is not intended to limit the invention in any way and the techniques described herein can be used to detect and identify any gene or mutation or polymorphism desired (e.g., polymorphisms or mutations associated with alcohol dependence, obesity, and cancer). 
     Once the primers are hybridized to the subject&#39;s DNA, a plurality of extension products having the marker or loci indicative of the trait are generated. Preferably, the extension products are generated through a polymerase-driven amplification reaction, such as multiplex PCR or multiplex Ligase Chain Reaction (LCR). In some embodiments, one or more fluorescent labels are employed. That is, by some methods, individual extension products are generated by PCR in the presence of different fluorescent labels so that the resulting extension products are fluoresce at different wavelengths (e.g., different colors are seen for each individual extension product on a detector). These embodiments facilitate the analysis of multiple patient samples in the same assay or multiple markers on the same or different genes. The extension products are then pooled according to similar melting behaviors and then the pooled samples are separated on the basis of melting behavior (e.g., TTGE or DHPLC). 
     In some approaches, for example, the extension products are isolated from the reactants in the amplification reaction, suspended in a non-denaturing loading buffer, and are loaded on a TTGE denaturing gel (e.g., an 8%, 7M urea polyacrylamide gel). The sample can be heated to a temperature sufficient to denature a DNA duplex and then cooled to a temperature that allows reannealing, prior to suspending the DNA in the non-denaturing loading buffer. The extension products are then loaded into a single lane or multiple lanes, as desired. Next, an electrical current is applied to the gel and extension products. 
     Subsequently, the temperature of the denaturing gel is gradually raised, while maintaining the electrical current, so as to separate the extension products on the basis of their melting behaviors. Once the fragments have been separated by size and melting behavior, one can identify the presence or absence of mutations or polymorphisms at the screened loci by analyzing the migration behavior of the extension products. By employing the fluorescent labels above, one can rapidly identify the differing extension products or patient samples, as well. 
     In other approaches, the extension products are isolated from the reactants and suspended in a DHPLC buffer (e.g., 0.1M TEAA pH 7.0). The extension products are then injected onto a DHPLC column (e.g., an ion-pair reverse phase HPLC column composed of alkylated non-porous poly(styrene-divinylbenzene)) that has been equilibrated to an appropriate denaturing temperature, depending on the size and composition of the DNA to be separated (e.g., 53° C. to 63° C.) in an appropriate buffer (e.g., 0.1 mM triethylamine acetate (TEAA) pH 7.0) and the extension products are allowed to bind. The presence of a denaturant (e.g., acetonitrile in TEAA) on the column is gradually raised over time so as to slowly elute the extension products from the column. Preferably a linear gradient is used. Presence of the extension products in the eluant is preferably accomplished using a UV detector (e.g., at 260 and/or 280 nm), however, greater sensitivity may be obtained using radioactivity, binding dyes, fluorescence or the techniques described in U.S. Pat. Nos. 5,795,976; 5,585,236; 6,024,878; 6,210,885; Huber, et al., Chromatographia 37:653 (1993); Huber, et al., Anal. Biochem. 212:351 (1993); Huber, et al., Anal. Chem. 67:578 (1995); and O&#39;Donovan et al., Genomics 52:44 (1998), which are all hereby incorporated by reference in their entireties including the references cited therein. 
     The appearance of a slower or faster migrating band at a temperature below or above the predicted melting point for the particular extension product in the TTGE approach, for example, indicates the presence of a mutation or polymorphism in the subject&#39;s DNA. Similarly, the appearance of a slower or faster eluting peak at a concentration of denaturant predicted to elute a wild-type or non-polymorphic homoduplex extension product in the DHPLC approach indicates the presence of a mutation or polymorphism in the subject&#39;s DNA. A heterozygous sample will display both homoduplex bands (wild-type homoduplexes and mutant homoduplexes), as well as, two heteroduplex bands that are the product of mutant/wild-type annealing. Because of base pair mismatches in these fragments, they melt significantly sooner than the two homoduplex bands. Accordingly, a user can rapidly identify the presence or absence of a mutation or polymorphism at the screened loci by either the TTGE or DHPLC approach and determine whether the tested subject has a predilection for a disease. 
     In a related embodiment, greater sensitivity is obtained by adding a “standard” DNA or “control” DNA to the DNA to be screened prior to amplification or after amplification, prior to separation of the DNA on the TTGE gel or DHPLC column. This insures the presence of heteroduplexes in the case of either a homozygous mutant, which normally would not display heteroduplexes, or a heterozygous mutant. Desired DNA standards include, but are not limited to, DNA that is wild-type for at least one of the traits that are being screened. Preferred standards include, but are not limited to, DNA that is wild-type for all of the traits that are being screened. A DNA standard can also be a mutant or polymorphic DNA. In some embodiments, particularly when the control DNA is added after amplification, the DNA standard is an extension product generated from a wild-type genomic DNA or a mutant genomic DNA. By this approach, the amplification phase of the method is performed as described above. That is, DNA from the subject to be screened and the DNA standard are hybridized with nucleic acid primers that flank regions of the genetic loci or markers that are associated with or linked to the traits being tested. In some embodiments, the DNA standard extension products are fluorescently labeled differently than the extension products generated from the screened samples so as to facilitate identification. 
     Extension products are then generated. If the subject being tested has at least one trait that is detected by the assay (e.g., a congenital disorder), then two populations of extension products are generated, a first population that corresponds to the standard DNA and a second population that corresponds to the subject&#39;s DNA having at least one mutation or polymorphism. Next, preferably, the two populations of extension products are isolated from the amplification reactants and are denatured by heat (e.g., 95° C. for 5 minutes), then are allowed to anneal by cooling (e.g., ice for 5 minutes). This ensures the formation of the heteroduplex bands in the presence of any relatively small mutation (e.g., point mutation, small insertion, or small deletion). The isolation and denaturing/annealing steps are not practiced with some embodiments, however. 
     Subsequently, by the TTGE approach, the two populations of extension products are suspended in a non-denaturing loading buffer and loaded on a denaturing polyacrylamide gel and separated on the basis of melting behavior, as described above. By the DHPLC approach, the two populations of extension products are suspended in a suitable buffer (e.g., 0.1M TEAA pH 7.0), loaded onto a buffer and temperature equilibrated DHPLC column and a linear gradient of denaturant is applied, as described above. Because the two populations of extension products are not perfectly complementary, they form heteroduplexes. Heteroduplexes are less stable than homoduplexes, have a lower melting temperature, and are easily differentiated from homoduplexes using the DNA separation techniques described above. One can identify the presence or absence of mutations or polymorphisms at the screened loci, for example, by comparing the migration behavior or elution behavior of the extension products generated from the screened DNA with the migration behavior or elution behavior of the DNA standard. If heteroduplexes are present, generally, two additional bands that correspond to the single extension product will appear on the gel or the extension products will elute from the column more rapidly than the control or standard DNA alerting the user to the presence of a mutation or polymorphism. Accordingly, a significant increase in sensitivity is obtained and a user can rapidly identify the presence or absence of a mutation or polymorphism in the tested DNA sample and, thereby, determine whether the screened subject has a predilection for a particular trait (e.g., a congenital disease). As stated above, by employing different fluorescent labels during individual amplification reactions, different fluorescently labeled extension products can be generated and the identification of particular markers can be facilitated. 
     Similarly, an increase in sensitivity can be obtained by mixing DNA from a plurality of subjects prior to amplification. Because the frequency of mutations or polymorphisms for most disorders are very low in the population, most of the extension products generated are wild-type DNA. Thus, most of the pool of DNA behaves as a DNA standard. That is, the predominant structure formed upon annealing after denaturation is a homoduplex, which can be rapidly distinguished from any heteroduplex that would appear if a subject were to have a polymorphism or mutation. Of course, extension products previously generated from multiple subjects can be used as control DNA by mixing the previously generated extension products with the extension products generated from the DNA that is being screened prior to electrophoresis. In several embodiments, the DNA from at least 2 subjects is mixed. Desirably, the DNA from at least 3 subjects is mixed. Preferably, the DNA from at least 4 subjects is mixed. It should be understood, however, that the DNA from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more subjects can be mixed prior to amplification or prior to separation on the basis of melting behavior, in accordance with some of the described embodiments. Again, by employing different fluorescent labels during individual amplification reactions, different fluorescently labeled extension products can be generated and the identification of genetic markers, in particular the same markers on different subjects (e.g., the amplification reactions for different subjects employ different fluorescent markers) can be facilitated. 
     In one embodiment, for example, DNA from a plurality of subjects to be tested is obtained by conventional methods, pooled, and hybridized with the desired nucleic acid primers. Extension products are then generated, as before. If at least one of the subjects being tested has at least one congenital disorder that is detected by the screen then two populations of extension products will be generated, a first population that corresponds to DNA from subjects that have the wild-type gene and a second population that corresponds to DNA from subjects having at least one mutant or polymorphic gene. 
     By one approach, the two populations of extension products are then isolated from the amplification reactants, suspended in a non-denaturing loading buffer, denatured by heat, annealed by cooling, and are separated by TTGE, as described above. By another approach, the two populations of extension products are isolated from the amplification reactants, suspended in a DHPLC loading buffer (0.1M TEAA pH 7.0), denatured by heat, annealed by cooling, and are separated on a DHPLC column, as described above. The presence of a subject in the DNA pool having at least one mutation or polymorphism is identified by analyzing the migration behavior of the DNA on the gel or the elution behavior from the column. The appearance of a slower or faster migrating band at a temperature below or above the predicted melting point for a particular extension product on the gel indicates the presence of a mutation or polymorphism in the DNA from one of the subjects. Similarly, the appearance of a slower or faster eluting extension product from the DHPLC column indicates the presence of a mutation or polymorphism in the DNA from one of the subjects. By repeating the analysis with smaller and smaller pools of samples, one can identify the individual(s) in the pool that has the mutation or polymorphism. Additionally, DNA standards can be used, as described above, to facilitate identification of the individual(s) having the mutation or polymorphism. Advantageously, some embodiments can be used to screen multiple samples at multiple loci that are on found on a plurality of genes in a single assay, thus, increasing sample throughput. The analysis of a plurality of DNA samples in the same assay also unexpectedly provides greater sensitivity. The section below describes a DNA separation technique that can be used with the embodiments described herein. 
     Multiple Extension Products of Similar Composition can be Separated on the Same Lane of a Denaturing Gel or in the Same Run on a DHPLC Column 
     It was discovered that multiple fragments of DNA, which vary slightly in length and/or composition, can be rapidly and efficiently resolved on the basis of melting behavior. Although the preferred methods for differentiating multiple fragments of DNA on the basis of melting behavior involve TTGE gel electrophoresis and DHPLC, it is contemplated that other conventional techniques that are amenable to DNA separation on the basis of melting behavior can be equivalently employed (e.g., size exclusion chromatography, ion exchange chromatography, and reverse phase chromatography on high pressure (e.g., HPLC), low pressure (e.g., FPLC), gravity-flow, or spin-columns, as well as, thin layer chromatography). 
     By one approach, a polyacrylamide gel having a porosity sufficient to resolve the DNA fragments on the basis of size (e.g., 4-20% acrylamide/bis acrylamide gel having a set concentration of denaturant) is used. The amount of denaturant in the gel (e.g., urea or formamide) can vary according to the length and composition of the DNA to be resolved. The concentration of urea in a polyacrylamide gel, for example, can be 3M, 3.5M, 4M, 4.5M, 5M, 5.5M, 6M, 6.5M, 7M, 7.5M, or 8M. In preferred embodiments, an 8% polyacrylamide gel with 7M urea is used. It should be emphasized, however, that other types of polyacrylamide gels, equivalents thereof, and agarose gels can be used. 
     The DNA samples to be resolved are placed in a non-denaturing buffer and can be loaded directly to the gel. In some embodiments, for example, when heteroduplex formation is desired to increase the sensitivity of the assay, it is desirable to heat the double stranded DNA to a temperature that permits denaturation (e.g., 95° C. for 5-10 minutes) and then slowly cool the DNA to a temperature that allows annealing (e.g., ice for 5-10 minutes) prior to mixing with the loading buffer. Preferably, the DNA is loaded onto the gel in a total volume of 10-20 μl. Preferably, a Temporal Temperature Gradient Gel Electrophoresis (TTGE) apparatus is used. A commercially available system that is suitable for this technique can be obtained from BioRad. The gel can be run at 120, 130, 140, 150, 175, 200, 220, 250, 275, or 300 V for 1.5-10 hours, for example. 
     Once the DNA has been loaded, an electrical current is applied to begin separating the fragments on the bass of size and the temperature of the gel is raised gradually. In one embodiment, for example, the melting behavior separation is performed by raising the temperature beyond 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., or 75° C. at approximately 5.0 C.°/hour-0.5° C./hour in 0.1° C. increments. 
     Once the extension products have been separated by melting behavior, the gel can be stained to reveal the separated DNA. Many conventional stains are suitable for this purpose including, but not limited to, ethidium bromide stain (e.g., 1% ethidium bromide in a 1.25×Tris Acetate EDTA pH 8.0 (TAE) solution), fluorescent stains, silver stains, and colloidal gold stains. In some embodiments, it is desirable to destain the gel (e.g., 20 minutes in a 1.25×TAE solution). After staining, the gel can be analyzed visually (e.g., under a U.V. lamp) and/or with a digital camera and computer software such as, the Eagle Eye System by Stratagene or the Gel Documentation System (BioRad). Additionally, when fluorescent markers are employed, conventional detectors that emit various wavelengths of light can be used so as to identify the presence and position of separated fluorescently labeled extension products. 
     Mutations or polymorphisms are easily identified by comparing the migration behavior of the DNA to be screened with the migration behavior of a control DNA and/or by monitoring the melting temperature of the extension products generated from the screened DNA. Desirable “control” DNA or “standard” DNA includes a DNA that is wild-type or non-polymorphic for at least one loci that is screened and preferred standard DNA is wild-type or non-polymorphic for all of the loci that are being screened. Because this DNA separation technique is sufficiently sensitive to identify a single base pair substitution in a DNA fragment up to 600 base pairs in length, small changes in the melting behaviors and migration of the extension products can be rapidly identified. The standard or control DNA can also be fluorescently labeled (preferably with a fluorescent label that is different than the one employed for the screened samples) to facilitate the analysis. 
     By another approach, DHPLC is used to resolve heteroduplex and homoduplex molecules of several PCR extension products in a single assay. Preferably, the heteroduplex and homoduplex extension products are separated from each other by ion-pair reverse phase high performance liquid chromatography. In one embodiment, a DHPLC column that contains alkylated non-porous poly(styrene-divinylbenzene) is used. Preferably, the DHPLC column is equilibrated in an appropriate degassed buffer, referred to as Buffer “A” (e.g., 0.1M TEAA pH 7.0) and is kept at a constant temperature somewhat below the predicted melting temperature of the extension products (e.g., 53° C.-60° C., preferably 50° C.). A plurality of extension products that may be generated from a plurality of different loci, as described herein, are suspended in Buffer A and are injected onto the DHPLC column. The Buffer A is then allowed to run through the column for a time sufficient to insure that the extension products have adequately bound to the column. Preferably, flow rate and the amount of gas (e.g., argon or helium) are adjusted and kept constant so that the pressure on the column does not exceed the recommended level. Gradually, degassed denaturing buffer, referred to as Buffer “B”, (e.g., 0.1M TEAA pH 7.0 and 25% acetonitrile) is applied to the column. Although an isocratic gradient can be used, a gradual linear gradient is preferred. By one approach, to separate fragments that range in size from 200-450 bp, for example, a gradient of 50%-65% Buffer B (0.1M TEAA pH 7.0 and 25% acetonitrile) is used. Of course, as the size of extension products to be separated on the DHPLC column decreases, the gradient and/or the amount of denaturant in Buffer B can be reduced, whereas, as the size of extension products to be separated on the DHPLC column increases, the gradient and/or the amount of denaturant in Buffer B can be increased. 
     The DHPLC column is designed such that double stranded DNA binds well but as the extension products become partially denatured the affinity to the column is reduced until a point is reached at which the particular extension product can no longer adhere to the column matrix. Typically, heteroduplexes denature before homoduplexes, thus, they would be expected to elute more rapidly from the column than homoduplexes. 
     In some embodiments, particularly embodiments concerning the separation of a plurality of different extension products (e.g., extension products generated from a plurality of loci), the choice of primers and, thus, the extension products generated therefrom, requires careful design. For example, a GC-clamp or other artificial sequence can be used to adjust the melting characteristics and increase the length of a particular DNA fragment, if needed, to facilitate separation on the DHPLC or improve resolution of the extension products. By one approach, each set of primers in a multiplex reaction are designed and selected to generate an extension product that has a unique homoduplex and heteroduplex elution behavior. In this manner, each species can be easily identified. 
     By another approach, each set of primers are designed to generate extension products that have homoduplexes with very similar melting characteristics. By this strategy, all of the homoduplexes will elute at the same or very similar concentration of denaturant, which is different than the concentration of denaturant required to elute the heteroduplexes. Accordingly, the elution of a species of extension product outside of the expected range for the homoduplexes indicates the presence of a mutation or polymorphism. 
     In the case that the extension products happen to have overlapping retention times/elution behaviors, the DHPLC conditions can be adjusted to include a primary separation on the basis of size prior to increasing the concentration of the denaturant on the column to improve resolution. The techniques described in Huber, et al., Anal. Chem. 67:578 (1995), hereby expressly incorporated by reference in its entirety, can be adapted for use with the novel DHPLC separation approach described herein. In one embodiment, for example, the alkylated non-porous poly(styrene-divinylbenzene) DHPLC column can be used to separate the extension products on the basis of size for a time sufficient to group the various populations of extension products (i.e., the homoduplexes and heteroduplexes generated from a single independent set of primers constitute a single population of extension products) prior to separating on the basis of melting behavior. 
     By one approach, the extension products are applied to the column, as above, in Buffer A and a shallow linear gradient of Buffer B (e.g., 30%-50% of a solution of 0.1M TEAA pH 7.0 and 25% acetonitrile for 200-450 by extension products) is applied so as to resolve the various populations of extension products. Then, a deeper linear gradient of Buffer B (e.g., 50%-65% of a solution of 0.1M TEAA pH 7.0 and 25% acetonitrile for 200-450 by extension products) is applied to resolve the homoduplexes from the heteroduplexes within each individual population of extension product. In this manner, the homoduplexes and heteroduplexes from each population of extension product can be resolved despite having overlapping elution behaviors. 
     It should be understood that the separation based on size can be performed at virtually any temperature as long as the extension products do not denature on the column, however, the amount of denaturant in Buffer B and the type of gradient may have to be adjusted. For example, the size separation can be accomplished at 4° C.-23° C., or 23° C.-40° C., or 40°-50° C., or 50° C.-60° C. Additionally, the size separation can be accomplished while the column is being gradually equilibrated to the temperature that is going to be used for the DHPLC. It should also be understood that the size separation can be performed on the same column with the appropriate gradient (shallow for a time sufficient to separate on the basis of size followed by a deeper gradient to separate on the basis of melting behavior). Additionally, columns in series can be used to separate extension products that have overlapping retention times/elution behaviors. For example, a first DHPLC column can be used to separate on the basis of size and a second DHPLC column can be used to separate on the basis melting behavior. 
     Mutations or polymorphisms are easily identified using the DHPLC techniques above by comparing the elution behavior of the DNA to be screened with the elution behavior of a control DNA. As above, desirable “control” DNA or “standard” DNA includes a DNA that is wild-type or non-polymorphic for at least one loci that is screened and preferred standard DNA is wild-type or non-polymorphic for all of the loci that are being screened. Control or standard DNA can also include extension products that are homoduplexes by virtue of a mutation or polymorphism or plurality of mutations or polymorphisms. Since the elution behavior of the wild type or non-polymorphic DNA or a homozygous mutant or polymorphism, represents the elution behavior of a homoduplex, one can use DHPLC values obtained from separating these controls, such as the retention time, elution time, or amount of denaturant required to elute the homoduplex as a basis for comparison to a screened sample to identify the presence of homoduplexes. Similarly, a control DNA can be a known heteroduplex and the elution behavior values described above can be used to identify the presence of a heteroduplex in a screened sample. 
     Additionally, the separated extension products can be collected after passing through the DHPLC column or TTGE gel or reamplified and sequenced to verify the existence of the mutation or polymorphism. Further, the identified products can be isolated from the gel and sequenced. Sequencing can be performed using the conventional dideoxy approach (e.g., Sequenase kit) or an automated sequencer. Preferably, all possible mutant fragments are sequenced using the CEQ 2000 automated sequencer from Beckman/Coulter and the accompanying analysis software. The mutations or polymorphisms identified by sequencing can be compiled along with the respective melting behaviors and the sizes of extension products. This data can be recorded in a database so as to generate a profile for each loci. 
     Additionally, this profile information can be recorded with other subject-specific information, for example family or medical history, so as to generate a subject profile. By creating such databases, individual mutations can be better characterized. Mutation analysis hardware and software can also be employed to aid in the identification of mutations or polymorphisms. For example, the “ALFexpress II DNA Analysis System”, available from Amersham Pharmacia Biotech and the “Mutation Analyser 1.01”, also available from Amersham Pharmacia Biotech, can be used. Mutation Analyser automatically detects mutations in sample sequence data, generated by the ALFexpress II DNA analysis instrument. The section below describes embodiments that allow for the identification of a mutation or polymorphism at multiple loci in a plurality of genes in a single assay. 
     Identification of the Presence or Absence of a Mutation or Polymorphism at Multiple Loci in a Plurality of Genes in a Single Assay 
     The DNA separation techniques described herein can be used to rapidly identify the presence or absence of a mutation or polymorphism at multiple loci in a plurality of genes in a single assay (e.g., in a single reaction vessel or multiple reaction vessels). Accordingly, a biological sample containing DNA is obtained from a subject and the DNA is isolated by conventional means. For some applications, it may be desired to screen the RNA of a subject for the presence of a genetic disorder (e.g., a congenital disease that arises through a splicing defect). In this case, a biological sample containing RNA is obtained, the RNA is isolated, and then is converted to cDNA by methods well known to those of skill in the art. DNA from a subject or cDNA synthesized from the mRNA obtained from a subject can be easily and efficiently isolated by various techniques known in the art. Also known in the art is the ability to amplify DNA fragments from whole cells, which can also be used with the embodiments described herein. Thus, the DNA sample for use with the embodiments described herein need only be isolated in the sense that the DNA is in a form that allows for PCR amplification. 
     In some embodiments, genomic DNA is isolated from a biological sample by using the Amersham Pharmacia Biotech “GenomicPrep Blood DNA Isolation Kit”. The isolation procedure involves four steps: (1) cell lysis (cells are lysed using an anionic detergent in the presence of a DNA preservative, which limits the activity of endogenous and exogenous Dnases); (2) RNAse treatment (contaminating RNA is removed by treatment with RNase A); (3) protein removal (cytoplasmic and nuclear proteins are removed by salt precipitation); and (4) DNA precipitation (genomic DNA is isolated by alcohol precipitation). EXAMPLE 1 also describes an approach that was used to isolate DNA from human blood. 
     Once the sample DNA has been obtained, primers that flank the desired loci to be screened are designed and manufactured. Preferably, optimal primers and optimal primer concentrations are used. Desirably, the concentrations of reagents, as well as, the parameters of the thermal cycling are optimized by performing routine amplifications using control templates. Primers can be made by any conventional DNA synthesizer or are commercially available. Optimal primers desirably reduce non-specific annealing during amplification and also generate extension products that resolve reproducibly on the basis of size or melting behavior and, preferably, both. Preferably, the primers are designed to hybridize to sample DNA at regions that flank loci that can be used to diagnose a trait, such as a congenital disease (e.g., loci that have mutations or polymorphisms that indicate a human disease). 
     Desirably, the primers are designed to detect loci that diagnose conditions selected from the group consisting of familial hypercholesterolemia (FH), cystic fibrosis, Tay-sachs, thalassemia, sickle cell disease, phenylketonuria, galactosemia, fragile X syndrome, hemophilia A, myotonic dystrophy, medium-chain acyl CoA dehydrogenase, maturity onset diabetes, cystinuria, methylmolonic acidemia, urea cycle disorders, hereditary fructose intolerance, hereditary hemachromatosis, neonatal thrombocytopenia, Gaucher&#39;s disease, tyrosinemia, Wilson&#39;s disease, alcaptonuria, hypolactasia, Baker&#39;s disease, argininemia Adenomatous polyposis coli (APC), Adult Polycystic Kidney disease, a-1-antitrypsin deficiency, Duchenne Muscular Dystrophy, Hemophilia A, Hereditary Nonpolyposis colorectal cancer, Huntingtons disease, Marfans syndrome, Myotonic dystrophy, Neurofibromatosis, Osteogenesis imperfecta, Retinoblastoma, Sickle cell disease, Freidrichs ataxia, Hemoglobinopathies, Leber&#39;s hereditary optic neuropathy, MCAD, Canavan&#39;s disease, Retintitus Pigmentosa, Bloom Syndrome, Fanconi anemia, and Neimann Pick disease. Preferably, the primers are designed to detect the presence or absence of polymorphisms or mutation associated with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). Primers can be designed to amplify any region of DNA, however, including those regions known to be associated with diseases such as alcohol dependence, obesity, and cancer. It should be understood that the embodiments described herein can be used to detect any gene, mutation, or polymorphism found in plants, virus, molds, yeast, bacteria, and animals. 
     Preferred primers are designed and manufactured to have a GC rich “clamp” at one end of a primer, which allows the dsDNA to denature in a “zipper-like” fashion. As one of skill will appreciate, PCR requires a “primer set”, which includes a first and a second primer, only one of which has the GC clamp so as to allow for separation of the double stranded molecule from one end only. Since the GC clamp is significantly stable, the rest of the fragment melts but does not completely separate until a point after the inflection point of the DNA, which contains the mutation or polymorphism of interest. The denaturant in the gel or on the column allows the temperature of melting to be lower and allows the inflection point of the melt to be longer in terms of temperature and, thus, the sensitivity to temperature at the inflection point is less (i.e., increment temperature=less increment melting), which increases the resolution. 
     Additionally, desirable primers are designed with a properly placed GC-clamp so that extension products that contain a single melting domain are produced. Preferably, the primers are selected to complement regions of introns that flank exons containing the genetic markers of interest so that polymorphisms or mutations that reside within the early portions of exons are not masked by the GC clamp. For example, it was discovered that GC clamps significantly perturb melting behavior and can prevent the detection of a polymorphism or mutation by melting behavior if the mutation or polymorphism resides too close to the GC clamp (e.g., within 40 nucleotides). By performing amplification reactions with control templates, optimal primer design and optimal concentration can be determined. The use of computer software, including, but not limited to, WinMelt or MacMelt (Bio-Rad) and Primer Premire 5.0 can aid in the creation and optimization of primers and proper positioning of the GC-clamp. Accordingly, many of the primers and groupings of primers described herein, as used in a particular assay (e.g., to screen for HNPCC) are embodiments of the invention. EXAMPLE 2 further describes the design and optimization of primers that allowed for the high-throughput multiplex PCR technique described herein. 
     Once optimal primers are designed and selected, the DNA sample is screened using the inventive multiplex PCR technique. In some embodiments, for example, approximately 25 ng-500 ng of template DNA (preferably, 200 ng for human genomic DNA) is suspended in a buffer comprising: 10 mM Tris (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTPs, 50 pmol of each primer, and 1 unit Taq polymerase per primer set in a total volume of 50 μl. Preferably, amplification is performed under the same conditions that were used to design the primers. In some embodiments, for example, amplification is performed on a conventional thermal cycler for 30 cycles, wherein each cycle is: 1 minute @ 95° C., 58° C. for 1 minute, 72° C. for 1 minute. Final extension is performed at 72° C. for 5 minutes. When the primers have a GC clamp, it was found that conditions often favor an amplification reaction having over 40 cycles, wherein each cycle is: 35 seconds @ 95° C., 120 seconds @ 50-57° C., and 60 seconds+3 seconds/cycle @ 72° C. Thermal cyclers are available from a number of scientific suppliers and most are suitable for the embodiments described herein. 
     Once the PCR reaction is complete, the extension products are desirably isolated by centrifugal microfiltration using a standard PCR cleanup cartridge, for example, Qiagen&#39;s QIAquick 96 PCR Purification Kit, according to manufacture&#39;s instructions. Isolation or purification of the extension products is not necessary to practice the invention, however. The isolated extension products can then be suspended in a non-denaturing loading buffer and either loaded directly on a DHPLC column or TTGE denaturing gel. The sample can also be denatured by heating (e.g., 95° C. for 5-10 minutes) and annealed by cooling (e.g., ice for 5-10 minutes) prior to loading onto the DHPLC column or TTGE denaturing gel. The various extension products are then separated on a TTGE denaturing gel or DHPLC column on the basis of melting behavior, as described above and, after separation, the extension products can be analyzed for the presence or absence of polymorphisms or mutations. EXAMPLES 3 and 4 describe experiments that verified that multiple loci on a plurality of genes can be screened in a single assay. The section below describes a method of genetic analysis, wherein improved sensitivity of detection was obtained by adding a DNA standard to the screened DNA. 
     Improved Sensitivity was Obtained Wizen a DNA Standard was Mixed with the Screened DNA 
     It was also discovered that greater sensitivity in the inventive multiplex PCR reactions described herein can be obtained by mixing a DNA standard with the DNA to be tested prior to conducting amplification or after amplification but prior to separation on the basis of melting behavior. Desired DNA standards include, but are not limited to, DNA that is wild-type for at least one of the traits that are being screened and preferred DNA standards include, but are not limited to, DNA that is wild-type for all of the traits that are being screened. DNA standards can also be mutant or polymorphic DNA. In some embodiments, particularly when the control DNA is added after amplification, the DNA standard is an extension product generated from a wild-type genomic DNA or a mutant genomic DNA. Optionally, the control DNA can be labeled with a fluorescent label, which can be a label that is different than the fluorescent label used to label the extension products generated from the screened sample DNA. In this manner, the standard or control DNA is easily differentiated from the DNA that is being screened. 
     By one approach, the DNA from the subject to be screened and the DNA standard are pooled and then the amplification reaction, as described above, is performed. Accordingly, optimal primers are designed and selected and approximately 25 ng-500 ng of template DNA (preferably, 200 ng for human genomic DNA) is suspended in a buffer comprising: 10 mM Tris (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTPs, 50 pmol of each primer, and 1 unit Taq polymerase per primer set in a total volume of 50 μl. Preferably, amplification is performed under the same conditions that were used to design the primers. In some embodiments, amplification is performed on a conventional thermal cycler for 30 cycles, wherein each cycle is: 1 minute @ 95° C., 58° C. for 1 minute, 72° C. for 1 minute. Final extension is performed at 72° C. for 5 minutes. When the primers have a GC clamp, however, conditions often favor an amplification reaction having over 40 cycles, wherein each cycle is: 35 seconds @ 95° C., 120 seconds @ 50-57° C., and 60 seconds+3 seconds/cycle @ 72° C. 
     If the subject being tested has at least one disorder that is detected by the assay then two populations of extension products are generated, a first population that corresponds to the standard DNA and a second population that corresponds to the subject&#39;s DNA having at least one mutation or polymorphism. The pool of extension products are desirably isolated from the amplification reactants, as above, and are suspended in a non-denaturing loading buffer. Preferably, the extension products are then denatured by heat (e.g., 95° C. for 5 minutes), and are allowed to anneal by cooling (e.g., ice for 5 minutes) prior to loading on the TTGE denaturing gel or DHPLC column. In this manner, the formation of heteroduplexes will be favored if the subject has a mutation or polymorphism because the two populations of extension products are not perfectly complementary. However, the isolation and denaturing/annealing steps are not necessary for some embodiments. 
     By another approach, the DNA standard is added to the extension products generated from the tested subject&#39;s DNA after the amplification reaction. As above, the pooled DNA sample is preferably denatured by heat (e.g., 95° C. for 5 minutes), and allowed to anneal by cooling (e.g., ice for 5 minutes). This second approach also produces heteroduplexes if the extension product and the DNA standard are not perfectly complementary. 
     Next, the TTGE denaturing gel or DHPLC column is loaded and the extension products are separated on the basis of melting behavior, as described above. Since heteroduplexes are less stable than homoduplexes and have a lower melting temperature, the presence or absence of a mutation or polymorphism in the tested DNA sample is easily determined. By comparing the migration behavior or elution behavior of the extension products generated from the screened DNA with the migration behavior of the DNA standard, a user can rapidly determine the presence or absence of a mutation or polymorphism (e.g., two additional bands that correspond to the single extension product will appear on the gel when a mutation or polymorphism is present in the tested DNA or a population of extension products will elute from the DHPLC column earlier than homoduplex controls or the majority of homoduplexes present in the sample). The section below describes a method of genetic analysis, wherein improved efficiency and sensitivity of detection was obtained by screening multiple DNA samples in the same assay. 
     Improved Sensitivity was Obtained when Multiple DNA Samples were Screened in the Same Assay 
     It was also discovered that an improved sensitivity of detection and increased throughput could be obtained by mixing DNA from a plurality of subjects prior to amplification. Because the frequency of mutations or polymorphisms for most disorders are very low in the population, most of the extension products generated correspond to wild-type or non-polymorphic DNA. Accordingly, most of the DNA in a reaction comprising DNA from a plurality of subjects behave similar to a DNA standard. That is, the predominant structure formed upon annealing after denaturation is a homoduplex, which can be rapidly distinguished from any heteroduplex that would appear if a subject were to have a mutation or polymorphism. Although the reaction is “dirty” from the perspective that the identity of each subject&#39;s DNA is not known initially, the identity of any polymorphic or mutant DNA can be determined through a process of elimination. For example, by repeating the analysis with smaller and smaller pools of samples, one can identify the individual(s) in the pool that have the mutation or polymorphism. Additionally, DNA standards can be used, as described above, to facilitate identification of the individual(s) having the mutation or polymorphism. Optionally, the each DNA can be labeled with a different fluorescent label so that identification of the variant is easily determined. 
     By one approach, DNA from a plurality of subjects to be tested is obtained by conventional methods, pooled, and hybridized with the desired nucleic acid primers. Accordingly, optimal primers are designed and selected and approximately 25 ng-500 ng of template DNA (preferably, 200 ng for human genomic DNA) is suspended in a buffer comprising: 10 mM Tris (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTPs, 50 pmol of each primer, and 1 unit Taq polymerase per primer set in a total volume of 50 μl. Preferably, amplification is performed under the same conditions that were used to design the primers. In some embodiments, amplification is performed on a conventional thermal cycler for 30 cycles, wherein each cycle is: 1 minute @ 95° C., 58° C. for 1 minute, 72° C. for 1 minute. Final extension is performed at 72° C. for 5 minutes. When the primers have a GC clamp, however, conditions often favor an amplification reaction having over 40 cycles, wherein each cycle is: 35 seconds @ 95° C., 120 seconds @ 50-57° C., and 60 seconds+3 seconds/cycle @ 72° C. 
     The pool of extension products are preferably isolated from the amplification reactants, as above, and are suspended in a non-denaturing loading buffer. Preferably, the extension products are then denatured by heat (e.g., 95° C. for 5 minutes), and are allowed to anneal by cooling (e.g., ice for 5 minutes). In this manner, the formation of heteroduplexes will be favored if the subject has a mutation or polymorphism because the two types of extension products are not perfectly complementary. Again, the isolation and denaturing/annealing steps are not performed in some embodiments and fluorescent labels can be employed. 
     Next, the TTGE denaturing gel or DHPLC column is loaded and the extension products are separated on the basis of melting behavior, as described above. When one of the subjects being tested has at least one trait that is detected by the screen, heteroduplexes are detected on the gel or eluting from the DHPLC column. The assay can be then repeated with smaller pools of samples and assays with a DNA standard can be conducted with individual samples to confirm the identity of the subject having the mutation or polymorphism. EXAMPLE 5 describes an experiment that verified that an improved sensitivity can be obtained by mixing a plurality of DNA samples. EXAMPLE 6 describes an experiment that verified that multiple genes and multiple loci therein can be screened in a plurality of subjects, in a single assay. EXAMPLE 7 describes the screening of multiple genes and multiple loci therein, in a plurality of subjects, in a single assay using a DHPLC approach. The section below describes the optimization of primer design in the context of an approach that was used to detect mutations and/or polymorphisms in the CFTR gene. 
     Optimization of Primer Design and Extension Product Design Facilitates Identification of Genetic Markers Associated with HNPCC 
     Using the approaches detailed in the previous sections, a preferred embodiment concerns the identification of the presence or absence of genetic markers, mutations, or polymorphisms that are associated with HNPCC. The sequences of genes associated with HNPCC can be found in U.S. Pat. Nos. 5,922,855; 6,165,713; 6,191,268; 6,538,108 and U.S. patent application Ser. Nos. 08/209,521 and 08/154,792, all of which are hereby expressly incorporated by reference in their entireties. 
     By one approach, almost the entire coding sequences for the mismatch repair genes mutL homolog 1 (MLH1) and mutS homologue 2 (MSH2) are scanned for the presence or absence of genetic markers, mutations, or polymorphisms that contribute to HNPCC. (See EXAMPLE 8). TABLE A provides the sequences of exons of the MLH1 and MSH2 genes and several oligonucleotide primers that have been used to screen regions of these genes for the presence or absence of genetic markers, polymorphisms, and mutations that are associated with HNPCC. Where indicated, the notation (*) refers to a GC clamp, an additional non-genetic GC rich sequence that is added to one of the two primers in a pair to add stability to the PCR product, as explained above and in Example 2 below. TABLE B also lists many oligonucleotide primers that have been used to screen regions of the MLH1 and MSH2 genes for the presence or absence of genetic markers, polymorphisms, and mutations that are associated with HNPCC. TABLE B also shows the starting and ending point for each primer as it relates to the publicly available gene sequence for the MLH1 and MSH2 genes (GenBank Accession NoS. AY217549 and NM000251, the contents of which are expressly incorporated by reference in its entirety). It is contemplated that primers that are any number between 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides upstream or downstream of the primers identified in TABLE A or B can be used with embodiments of the invention so long as these primers produce extension products that melt over long stretches of DNA (approximately 25, 50, 75, 100, 125, or 150 nucleotides) at approximately the same temperature (within 0° C.-1.5° C.) and are resolvable on a TTGE gel or DHPLC column. 
     As detailed above, the sequences of the MLH1 and MSH2 genes are readily available. Accordingly, embodiments include methods of diagnosing HNPCC with primers that are any number from 1-75 nucleotides upstream or down stream from the beginning or ending of the primers listed in TABLE A or B, preferably using the approaches described herein. It is also preferred that said methods use primers that produce extension products that melt over long stretches of DNA (approximately 25, 50, 75, 100, 125, or 150 nucleotides) at approximately the same temperature (within 0° C.-1.5° C.) and are resolvable on a TTGE gel or DHPLC column. Preferably, these extension products are obtained, grouped, and separated as described below. 
     By one approach, samples of DNA were obtained from several subjects to be screened using the approaches described herein and were disposed in a plurality of 96-well micro-titer plates such that a single row of each plate corresponded to a single tested subject. In some cases, 7 total plates were used per assay, wherein each plate has 7 sample lanes (i.e., 7 subjects analyzed) and an eighth lane was used for positive control sample DNA. Amplification buffer, amplification enzyme (e.g., Taq polymerase), and DNTPs were added to the sample DNA in each well, as described above, and a plurality of primer sets that encompass most of the gene (e.g., 84 primer sets) were to yield a final volume of 10 μl. The primer sets that were employed in a first set of tests are identified in TABLE A. TABLE C describes the plate setup for these amplification reactions as well as a protocol for PCR reactions, whereas TABLE D describes the conditions for the TTGE separation for these tests and describes the groupings for the various fragments for TTGE separation. Preferred methods of diagnosing HNPCC employ the primers of TABLE A to generate extension products that are grouped according to TABLE D and separated by melting behavior (e.g., TTGE). By using this approach, a rapid, inexpensive, and efficient diagnosis of the presence or absence of a marker associated with HNPCC can be ascertained. The names of the extension products, “fragments” in TABLE C and TABLE D correspond to the names of the primer sets used throughout. The top line numbering on the master plate chart of TABLE C refers to the location of the well on the 96 well plate, the “MLH stack” or “MSH stack” of TABLE D refers to the grouping pool of the extension products prior to TTGE and the alternating shaded and unshaded sections of TABLE D show grouping pools of extension products that can be run under the same TTGE conditions (which are shown under “Run group”). 
     Although multiplex PCR reactions can be employed, preferably, each primer set is run in an individual reaction. Conditions for PCR were, in one case for example: 5 minutes at 96° C. for initial denaturing followed by 35 total cycles of: 30 seconds at 94° C. and 30 seconds at the annealing temperature or at a gradient of 49° C. to 63° C. and a final 10 minutes at 72° C. to complete synthesis of any partial products. Most preferred are primers that have an annealing temperature between 49° C. and 63° C., though many of the primer sets have annealing temperatures that are at 49° C., 52° C., 59° C., and 62.4° C. An approximately 3° C. window is allowed for each plate (e.g., primers having annealing temperatures that are within 3° C. of one another are grouped on a single plate). Programs such as WINMELT were used to determine whether the primers could be grouped into various primer sets that have similar annealing temperatures so that individual groups of primers can be amplified by Polymerase Chain Reaction (PCR) on the same plate. 
     Once the extension products had been generated they were grouped, pooled, and mixed with loading dye. Eight Multi G groups (Multi-Grouping pools of extension products) were used for the extension products “fragments” generated by the various primer sets, which belong to one of the eight groups are identified in TABLE C and TABLE D (some of the run groups on the separate MLH1 and MSH2 Table have identical conditions). After grouping and pooling, the samples were loaded onto a TTGE gel. TABLE D also lists the start and stop temperatures for the TTGE, for each Multi G group, under ‘run conditions’. Preferably, the TTGE is run with a very shallow temperature gradient, e.g., about 1.0° C./hour for a total of three hours, at high voltage, e.g., 150 volts. Once the separation was complete, the gels were grouped, stained with ethidum bromide, and analyzed by the Decode system. The analysis above was rapid, inexpensive, and very effective at detecting mutations and/or polymorphisms, many of which go undetected or are not analyzed by others in the field. 
     Whereas many in the field seek to design primers that optimally anneal with a template DNA, it has been discovered that primers can also be designed to produce an optimal extension product (e.g., a fragment of short length with a reliable and rapid melting point). Preferably, primers are designed to generate extension products that are approximately 100-300 nucleotides in length and that have long stretches of DNA that melt at approximately the same temperature (e.g., DNA stretches that are 25, 35, 45, 55, 65, 75, 85, 95, 100, 125, 15, 175, or 200 nucleotides that melt at the same temperature or within about a 0° C. to about a 1.5° C. temperature difference). 
     Programs such as WINMELT were used to evaluate the melting behavior of extension products generated from the various primer sets and test TTGE separation of the extension products generated by the various primer sets were also performed to ensure that the predicted melting behavior was represented on the gel. For example,  FIGS. 1-4  show graphs of four extension products produced by two primer sets that amplify portions of the cystic fibrosis gene (CTFR). The flat melting curve shown in these figures is preferred for the applications described herein because the extension products melt rapidly and are quickly retarded in the gel, which improves resolution and allows multiple different extension products to be separated in the same lane on a TTGE gel. That is, by grouping extension products that have flat melting profiles, which are within, approximately 1.5° C. of one another, it allows a shallow TTGE temperature ramp (e.g., 1° C. change per hour for 3 hours) or shallow DHPLC temperature ramp, which increases the sensitivity, allowing multiple extension products to be separated in the same lane, which increases throughput and reduces the cost of the analysis. 
     By analogy, TABLE D shows several of the characteristics of the extension products generated by the primers described herein. In particular, the PCR annealing temperature for the primer set used to generate the extension product (“PCR temp.”) is provided. Further, the Multi 
     G/stack group is also listed. The following examples describe the foregoing methodologies in greater detail. The first example describes an approach that was used to isolate DNA from human blood. 
     Example 1 
     A sample of blood was obtained from a subject to be tested by phlebotomy. A portion of the sample (e.g., approximately 1.0 ml) was added to approximately three times the sample volume or 3.0 ml of a lysis solution (10 mM KHCO 3 , 155 mM NH 4 Cl, 0.1 mM EDTA) and was mixed gently. The lysis solution and blood were allowed to react for approximately five minutes. Next, the sample was&#39; centrifuged (×500 g) for approximately 2 minutes and the supernatant was removed. Some of the supernatant was left (e.g., on the walls of the vessel) to facilitate suspension. The pellet was then vortexed for approximately 5-10 seconds. An extraction solution, which contains chaotrope and detergent (Qiagen), was then added (e.g., 500 μl), the sample was vortexed again for approximately 5-10 seconds, and the solution was allowed to react for five minutes at room temperature. 
     Next, a GFX column, which are pre-packed columns containing a glass fiber matrix, was placed under vacuum (e.g., a Microplex 24 vacuum system) and the extracted solution containing the DNA was transferred to the column (e.g., in 500 μl aliquots). Once all of the sample has been passed through the column, the vacuum was allowed to continue for approximately 5 minutes. Subsequently, a wash solution (Tris-EDTA buffer in 80% ethanol) was added (e.g., approximately 500 μl) under vacuum. Once the wash solution had been drained from the column, the vacuum was allowed to continue for approximately 15 minutes. The GFX columns containing the DNA were then placed into sterile microfuge tubes but the lids were kept open. 
     Elution buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was then added to the column (e.g., approximately 100 μl of buffer that was heated to approximately 70° C.) and the buffer was allowed to react with the column for approximately 2 minutes. Then, the tubes containing the columns were centrifuged at ×5000 g for approximately 1.5 minutes. After centrifugation, the column was discarded and the microfuge tube containing the isolated DNA was stored at −20° C. The example below describes the design and optimization of primers that allowed for the inventive high-throughput multiplex PCR technique, described herein. 
     Example 2 
     Sets of primers for PCR amplification were designed for every exon of the following genes: Cystic Fibrosis Transmembrane Reductase (CFTR), Beta-hexosaminidase alpha chain (HEXA), PAH, Alpha globin-2 (HBA2), Beta globin (BBB), Glucocerebrosidase (GBA), Galactose-1-phosphae uridyl transferase (GALT), Medium chain acyl-CoA dehydrogenase (MCAD), Protease inhibitor 1 (PI), Factor VIII, FMR1, and Aspartoacylase (ASPA). The primers were designed from sequence information that was available from GenBank or from sequence information obtained from Ambry Genetics Corporation. Information regarding mutations or polymorphisms was obtained from The Human Gene Mutation Database. 
     One of the primers in each primer set contained a GC-clamp. It was discovered that the addition of a GC-clamp significantly altered the melting profile of the DNA extension product. Further, proper positioning of the GC-clamp served to level the melting profile. It was desired to position the GC-clamp so that a single melting domain across the fragment was created. Proper positioning of the GC-clamp was oftentimes needed to prevent the GC-clamp from masking the presence of a mutation or polymorphism (e.g., if the mutation or polymorphism is too close to the GC-clamp). Software was also used to optimize primer design. For example, many primers were designed with the aid of Primer Premiere 4.0 and 5.0 and appropriate positioning of the GC-clamps was determined using WinMelt software from BioRad. To maintain sensitivity of the test, the primers were designed to anneal at a minimum of 40 base pairs either upstream or downstream of the nearest known mutation in the intronic region of the genes. 
     Although multiplex PCR can be technically difficult when using the quantity of primers described herein, it was discovered that almost all of the PCR artifacts disappeared when salt concentration, temperature, primer selection, and primer concentration were carefully optimized. Optimization was determined for each primer set alone and in combination with other primer sets. Optimization experiments were conducted using Master Mix from Qiagen and a Thermocyler from MJ Research. The conditions for thermal cycling were 5 minutes @ 95° C. for the initial denaturation, then 30 cycles of: 30 seconds @ 94° C., 45 seconds @ 48-68° C., and 1 minute @ 72° C. A final extension was performed at 72° C. for 10 minutes. 
     In addition to primer compatibility, primers were selected to facilitate identification of extension products by electrophoresis. To optimize primer design in this regard, separate PCR reactions were conducted for each individual set of primers and the extension products were separated by the inventive DNA separation technique, described above. Identical parameters were maintained for each assay and the migration behavior for each extension product was analyzed (e.g., compared to a standard to determine a R f  value for each fragment). An R f  value is a unit less value that characterizes a fragment&#39;s mobility relative to a standard under set conditions. In many primer optimization experiments, for example, the generated extension products were compared to a standard extension product obtained from amplification of the first exon of the PAH (phenylalanine hydroxylase) gene. A measurement of the distance of migration of each band in comparison to the distance of migration of the first exon of PAH was recorded and the R f  value was calculated according to the following: 
     
       
         
           
             
               R 
               f 
             
             = 
             
               
                 
                   ( 
                   
                     migration 
                      
                     
                         
                     
                      
                     distance 
                      
                     
                         
                     
                      
                     of 
                      
                     
                         
                     
                      
                     fragment 
                   
                   ) 
                 
                  
                 
                     
                 
                  
                 cm 
               
               
                 
                   ( 
                   
                     migration 
                      
                     
                         
                     
                      
                     distance 
                      
                     
                         
                     
                      
                     of 
                      
                     
                         
                     
                      
                     PAH 
                      
                     
                         
                     
                      
                     exon 
                      
                     
                         
                     
                      
                     1 
                   
                   ) 
                 
                  
                 
                     
                 
                  
                 cm 
               
             
           
         
       
     
     By conducting these experiments, it was verified that the selected primers did not produce extension products that overlapped on the gel. Optimal primer selection was obtained when optimal PCR parameters were maintained and the extension products produced dissimilar R f  values. Finally, the multiplex PCR was tested with all sets of primers and it was verified that few artifacts were created during amplification. Embodiments of the invention include the primers provided in the Tables and sequence listing provided herein and methods of using said primers and/or groups of primers. The example below describes an experiment that verified that the embodiments described herein effectively screen multiple loci present on a plurality of genes in a single assay. 
     Example 3 
     Two independent PCR reactions were conducted to demonstrate that multiple loci on a plurality of genes can be screened in a single assay using an embodiment of the invention. In a first reaction, seven different loci from four different genes were screened and, in the second reaction, eight different loci from four different genes were screened. The primers used in each multiplex reaction are provided in Table 1. 
     
       
         
           
               
               
             
               
                 TABLE 1* 
               
               
                   
               
               
                 Multiplex #1 
                 Multiplex #2 
               
               
                   
               
             
            
               
                 Factor VIII 4 (SEQ. ID. Nos. 300 and 318) 
                 CFTR 23 (SEQ. ID. Nos. 296 and 314) 
               
               
                 Factor VIII 11 (SEQ. ID. Nos. 302 and 320) 
                 CFTR 18 (SEQ. ID. Nos. 295 and 313) 
               
               
                 Factor VIII 24 (SEQ. ID. Nos. 303 and 321) 
                 Factor VIII 11 (SEQ. ID. Nos. 302 and 320) 
               
               
                 PAH 9 (SEQ. ID. Nos. 311 and 329) 
                 Factor VIII 3 (SEQ. ID. Nos. 299 and 317) 
               
               
                 GBA 6 (SEQ. ID. Nos. 308 and 326) 
                 CFTR 24 (SEQ. ID. Nos. 330 and 331) 
               
               
                 Factor VIII 1 (SEQ. ID. Nos. 297 and 315) 
                 GBA 4 (SEQ. ID. Nos. 307 and 325) 
               
               
                 GALT 9 (SEQ. ID. Nos. 310 and 328) 
                 GALT 9 (SEQ. ID. Nos. 310 and 328) 
               
               
                   
                 GBA 3 (SEQ. ID. Nos. 306 and 324) 
               
               
                   
               
               
                 *Primers are stored in a 50 μM storage stock and a 12.5 μM working stock. 
               
               
                 Abbreviations are: Phenyl alanine hydroxylase (PAH), Glucocerebrosidase (GBA), Galactose-1- phosphate uridyl transferase (GALT), and cystic fibrosis transmembrane reductase (CFTR). The numbers following the abbreviations represent the exons probed. 
               
            
           
         
       
     
     The amplification was carried out in 25 μl reactions using a 2× Hot Start Master Mix, which contains Hot Start Taq DNA Polymerase, and a final concentration of 1.5 mM MgCl 2  and 200 μM of each dNTP (commercially available from Qiagen). In each reaction, 12.50 of Hot Start Master Mix was mixed with 1 of μlgenomic DNA (approximately 200 ng genomic DNA), which was purified from blood using a commercially available blood purification kit (Pharmacia or Amersham). Primers were then added to the mixture (0.5 μM final concentration of each primer). Then, ddH 2 O was added to bring the final volume to 25 μl. 
     Thermal cycling for the Multiplex #1 reaction was performed using the following parameters: 15 minutes @ 95° C. for 1 cycle; 30 seconds @ 94° C., 1 minute (4) 53° C., 1 minute and 30 seconds (4) 72° C. for 35 cycles; and 10 minutes @ 72° C. for 1 cycle. Thermal cycling for the Multiplex #2 reaction was performed using the following parameters: 15 minutes (4) 95° C. for 1 cycle; 30 seconds @ 94° C., 1 minute @49° C., 1 minute and 30 seconds @ 72° C. for 35 cycles; and 10 minutes @ 72° C. for 1 cycle. 
     After the amplification was finished, approximately 5 μl of each PCR product was mixed with 5 μlof non-denaturing gel loading dye (70% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 2 mM EDTA). The DNA in the two reactions was then separated on the basis of melting behavior on separate denaturing gels. Each gel was a 16×16 cm, 1 mm thick, 7M urea, 8% acrylamide/bis(37.5:1) gel composed in 1.25×TAE (50 mM Tris, 25 mM acetic acid, 1.25 mM EDTA). Separation was conducted for 4 hours at 150 V on the Dcode system (BioRad) and the temperature ranged from 51° C. to 63° C. with a temperature ramp rate of 3° C./hour. Subsequently, the gels were stained in 1 μg/ml ethidium bromide in 1.25×TAE for 3 minutes and destained in 1.25×TAE buffer for 20 minutes. The gels were then photographed using the Gel Doc 1000 system from BioRad. 
     The primers in Table 1 were selected and manufactured because they produced extension products with very different R f  values and the extension products were clearly resolved by separation on the basis of melting behavior. Although some bands were more visible than others on the gel, seven distinct bands were observed on the gel loaded with extension products generated from the Multiplex 1 reaction and eight distinct bands were observed on the gel loaded with extension products generated from the Multiplex 2 reaction. These results verified that the described method effectively screened multiple loci on a plurality of genes in a single assay. The example below describes another experiment that verified that the embodiments described herein can be used to effectively screen multiple loci present on a plurality of genes in a single assay. 
     Example 4 
     Experiments were conducted to differentiate extension products generated from wild-type DNA and extension products generated from mutant DNA. Samples of genomic DNA that had been previously identified to contain mutations or polymorphisms were purchased from Coriell Cell Repositories. The mutation or polymorphism that was analyzed in this experiment was the delta-F508 mutation of the CFTR gene. This mutation is a 3 by deletion in exon 10 of the CFTR gene. Other loci analyzed in these experiments included the Fragile X gene, exon 17; Fragile X gene, exon 3; Factor VIII gene exon 2; and the Factor VIII gene, exon 7. Both the known mutant and a control wild-type for CFTR exon 10 were amplified within a multiplex reaction and individually. PCR amplification was conducted as described in EXAMPLE 3; however, 0.25 μM (final concentration) of each primer was used. The primers used in these experiments were CFTR 10 (SEQ. ID. Nos. 294 and 312), FragX 17 (SEQ. ID. Nos. 305 and 323), FragX 3 (SEQ. ID. Nos. 304 and 322), Factor VIII 7 (SEQ. ID. Nos. 301 and 319) and Factor VIII 2 (SEQ. ID. Nos. 298 and 316). The numbers following the abbreviations represent the exons probed. 
     The DNA templates that were analyzed included known wild-type genomic DNA, known mutant genomic DNA, mixed wild-type genomic DNA from various subjects, and mixed wild-type and mutant genomic DNA. Approximately 200 ng of genomic DNA was added to each reaction. The mixed wild-type and mutant DNA sample had approximately 100 ng of each DNA type. Thermal cycling was carried out with a 15-minute. step at 95° C. to activate the Hot Start Polymerase, followed by 30 cycles of 30 seconds at @ 94 C, 1 minute at @ 53° C., 1 minute and 30 seconds at @ 72° C.; and 72° C. for 10 minutes. 
     After amplification, approximately 5 μl of the PCR product was mixed with 5 μl of non-denaturing gel loading dye (70% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 2 mM EDTA). The samples were then separated on a 16×16 cm, 1 mm thick, 6M urea, 6% acrylamide/bis (37.5:1) gel in 1.25×TAE (50 mM Tris, 25 mM acetic acid, 1.25 mM EDTA) for 5 hours at 130 V using the Dcode system (BioRad). The temperature ranged from 40° C. to 50° C. at a temperature ramp rate of 2° C./hour. The gels were then stained in 1 μg/ml ethidium bromide in 1.25×TAE for 3 minutes and destained in 1.25×TAE buffer for 20 minutes. The gels were then photographed using the Gel Doc 1000 system from BioRad. 
     The resulting gel revealed that the lane containing the extension products generated from the wild-type DNA using the CFTR10 primers had a mobility commensurate to the wild-type DNA standard, as did the extension products generated from the other primers and the wild-type DNA. That is, a single band appeared on the gel in these lanes. The lane containing the extension products generated from the template having the F508 mutant, on the other hand, showed 2 bands. One of the bands had the same mobility as the extension products generated from the wild-type or DNA standard and the other band migrated slightly faster. These two populations of bands represent the two populations of homoduplexes (i.e., wild-type/wild-type and mutant/mutant). The top band is the wild-type homoduplex and the lower band is the mutant F508 homoduplex. Similarly, the lane that contained the wild-type/mutant DNA mix exhibited two populations of extension products, one representing the wild-type homoduplex population and the other representing the mutant homoduplex. Since F508 is a 3 by deletion it failed to form heteroduplex bands in sufficient quantity to be visible on the gel. Thus, this experiment demonstrated that the described method effectively screened multiple genes, in a single assay, and detected the presence of a polymorphism in one of the screened genes. The example below describes an experiment that demonstrated that an improved sensitivity can be obtained by mixing a plurality of DNA samples. 
     Example 5 
     This example describes two experiments that verified that an improved sensitivity of detection can be obtained by (1) mixing the DNA samples from a plurality of subjects prior to amplification or by (2) mixing amplification products before separation on the basis of melting behavior. In these experiments, PCR amplifications of exon 9 of the GBA gene (Glucocerebrosidase gene) were used. DNA samples known to contain a mutation in exon 9 of the GBA gene were purchased from Coriell Cell Repositories. These DNA samples contain a homozygous mutation in exon 9 of the GBA gene (the N370S mutation). 
     In a first experiment, single amplification of exon 9 was performed in a 25 μl reaction. A Taq PCR Master Mix (containing Taq DNA Polymerase and a final concentration of 1.5 mM MgCl 2  and 200 μM dNTPs)(Qiagen) was mixed with 0.5 μM (final concentration) of primers (SEQ. ID. Nos. 309 and 327). The template genomic DNAs analyzed in this experiment included wild-type DNA, mutant DNA, and various mixtures of wild-type and mutant DNA. For the single non-mixed reactions, approximately 200 ng of genomic DNA was used for amplification. In the mixed samples, approximately 200 ng of DNA was again used, however, the percentage of wild-type to mutant genomic DNA varied. Thermal cycling was performed according to the following parameters: 10 minutes @ 94° C.; 30 cycles of 30 seconds @ 94° C., 1 minute@ 44.5° C., and 1 minute and 30 seconds @ 72° C.; and 10 minutes @ 72° C. 
     In the second experiment, the amplification products were mixed prior to separation on the basis of melting behavior. Amplification of both wild-type and mutant (N370S) exon 9 of the GBA gene was performed using 25 μl reactions, as before. The Taq Master Mix obtained from Qiagen was mixed with 200 ng of genomic DNA and 0.5 μM final concentration of both primers (SEQ. ID. Nos. 309 and 327). PCR was carried out for 30 cycles with an annealing temperature of 56° C. for 1 minute. The denaturation and elongation steps were 94° C. for 30 seconds and 72° C. for 1 minute and 30 seconds. Final elongation was carried out at 72° C. for 10 minutes. The extension products obtained from the single amplification of wild-type GBA exon 9 was then mixed with the extension products obtained from the single amplification of the mutant GBA exon 9. Next, the pooled DNA was subjected to denaturation at 95° C. for 10 minutes and cooled on ice for 5 minutes, then heated to 65° C. for 5 minutes and cooled to 4° C. This denaturation and annealing procedure was performed to facilitate the formation of heteroduplex DNA. 
     Once the extension products from both experiments were in hand, approximately 5 μl of both the prior to PCR mixture and post PCR mixture were loaded on 16×16 cm, 1 mm thick gels (7M Urea/8% acrylamide (37.5:1) gel in 1.25×TAE) using the gel loading dye and the Dcode system (BioRad), described above. The DNA on the gel was then separated at 150 V for 5 hours and the temperature was uniformly raised 2° C./hour throughout the run starting at 50° C. and ending at 60° C. 
     The gel was stained in 1 μg/ml ethidium bromide in 1.25×TAE buffer for 3 minutes and destained in buffer for 20 minutes. 
     It should be noted that the GBA gene has a pseudo gene, which was co-amplified by the procedure above. An extension product generated from this psuedo gene migrated slightly faster than the extension product generated from the true expressed gene on the gel. In all lanes, the band representing the extension product generated from the psuedo gene was present. Then next fastest band on the gel was the extension product generated from the GBA exon 9 wild-type allele. The extension product generated from the mutant GBA exon 9 allele comigrated with the wild-type allele and was virtually indistinguishable on the basis of melting behavior due to the single base difference. 
     The heteroduplexes formed in the mixed samples were easily differentiated from the homoduplexes. The samples mixed prior to PCR showed both homoduplexes (wild-type and mutant) along with heteroduplexes, which appeared higher on the gel. Thus, by mixing samples, either prior to amplification or prior to separation on the basis of melting behavior an improved sensitivity of detection was obtained. Since homoduplex bands no longer need to be resolved to identify a mutation or polymorphism, only the heteroduplex bands need to be resolved, the throughput of diagnostic analysis was greatly improved. The example below describes experiments that verified that the embodiments taught herein can be used to effectively screen multiple genes in a plurality of subjects, in a single assay, for the presence or absence of a polymorphism or mutation. 
     Example 6 
     Two experiments were conducted to verify that multiple genes from a plurality of subjects can be screened in a single assay for the presence or absence of a genetic marker (e.g. a polymorphism or mutation) that is indicative of disease. These experiments also demonstrated that an improved sensitivity of detection could be obtained by mixing DNA samples either prior to generation of extension products or prior to separation on the basis of melting behavior. 
     In both experiments, five different extension products were generated from three different genes in a single reaction vessel. The five different extension products were generated using the following primers: Factor VIII 1 (SEQ. ID. Nos. 297 and 315); GBA 9 (SEQ. ID. Nos. 309 and 327); GBA 11 (SEQ. ID. Nos. 332 and 333); GALT 5 (SEQ. ID. Nos. 334 and 335), and GALT 8 (SEQ. ID. Nos. 336 and 337). Abbreviations are: Glucocerebrosidase (GBA) and Galactose-1-phosphate uridyl transferase (GALT). The numbers following the abbreviations represent the exons probed. 
     Extension products were generated for each experiment in 25:1 amplification reactions using Qiagen&#39;s 2× Hot Start Master Mix (Contains Hot Start Taq DNA Polymerase, and a final concentration of 1.5 mM MgCl 2  and 200 :M of each dNTP). To each reaction, 12.5 μl of Hot Start Master Mix was added to 1 μl of genomic DNA (approximately 200 ng genomic DNA for the mutant DNA sample and the wild-type DNA sample), which was purified from human blood using Pharmacia Amersham Blood purification kits. For the experiment in which the DNA samples from a plurality of subjects were mixed prior to generation of the extension products, approximately 100 ng of wild-type genomic DNA was mixed with approximately 100 ng of mutant N370S genomic DNA. In both experiments, primers were added to achieve a final concentration of 0.5 :M for each primer and a final volume of 25 μl was obtained by adjusting the volume with ddH 2 O. 
     Thermal cycling for both experiments was performed using the following parameters: 15 minutes @ 95° C. for 1 cycle; 30 seconds @ 94° C., one minute @ 57° C., and one minute 30 seconds @ 72° C. for 35 cycles; and 10 minutes @ 72° C. for 1 cycle. After amplification, the extension products generated from the wild-type and mutant templates (the un-mixed samples) were separated from the PCR reactants using a PCR Clean Up kit (Qiagen). Then, approximately 10 μL of the wild-type and mutant DNA were removed from each tube and gently mixed in a single reaction vessel. This preparation was then denatured at 95° C. for 1 minute and rapidly cooled to 4° C. for 5 minutes. Finally, the preparation was brought to 65° C. for an additional 1.5 minutes. The extension products generated from the mixed sample (wild-type DNA and mutant DNA mixed prior to amplification) were stored until loaded onto a denaturing gel. 
     Next, approximately 10 μl of the unmixed sample was combined with 10 μl of loading dye and approximately 5:1 of the mixed sample was combined with 5:1 of loading dye. The loading dye was composed of 70% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, and 2 mM EDTA). The samples in loading dye were then loaded on separate 16×16 cm, 1 min thick, 7M urea, 8% acrylamide/bis(37.5:1) gels in 1.25×TAE (50 mM Tris, 25 mM acetic acid, 1.25 mM EDTA). The DNA was separated on the basis of melting behavior for 5 hours at 150 V on the Dcode system (BioRad). The temperature ranged from 56° C. to 68° C. at a temperature ramp rate of 2° C./hr. The gels were then stained in 1 μg/ml ethidium bromide in 1.25×TAE for 3 minutes and destained in 1.25×TAE buffer for 20 minutes. The gels were photographed using the Gel Doc 1000 system (BioRad). 
     In all lanes of the gel, 5 extension products generated from three different genes were visible in the following order from top to bottom: Factor VIII 1, GBA 9, GBA 11, GALT 8, and GALT 5. Two different extension products were generated from the GBA 9 primers, as described above. The less intense band below the homoduplex bands corresponded to an extension product generated from the pseudogene. In the lanes loaded with extension products generated from only the wild-type or mutant DNA template, it was difficult to distinguish the wild type homoduplex from the N370S mutant homoduplex. In the lane loaded with the extension products generated from the mixed DNA templates (wild-type and mutant DNA mixed prior to amplification) and the lane loaded with extension products (generated from wild type and mutant DNA separately) that were mixed after amplification, heteroduplex bands were easily visualized. These experiments verified that multiple genes can be screened in a plurality of individuals in a single assay and that a single nucleotide mutation or polymorphism can be detected. Further, these experiments demonstrate that screening a plurality of DNA samples in a single reaction vessel or adding a control DNA before or after amplification greatly improves the sensitivity of detection. By practicing the methods taught in this example, the throughput of diagnostic screening can be drastically improved and the cost of identifying genetic traits can be significantly reduced. The example below describes approaches to screen multiple genes in a plurality of subjects, in a single assay, for the presence or absence of a polymorphism or mutation using DHPLC. 
     Example 7 
     Multiple genes in a plurality of subjects, in a single assay, can be screened for the presence or absence of a polymorphism or mutation using a DHPLC separation approach. For example, five different extension products can be generated using the following primers: Factor VIII 1 (SEQ. ID. Nos. 297 and 315); GBA 9 (SEQ. ID. Nos. 309 and 327); GBA 11 (SEQ. ID. Nos. 332 and 333); GALT 5 (SEQ. ID. Nos. 334 and 335), and GALT 8 (SEQ. ID. Nos. 336 and 337). Abbreviations are: Glucocerebrosidase (GBA) and Galactose-1-phosphate uridyl transferase (GALT). The numbers following the abbreviations represent the exons probed. The extension products can be generated in 25:1 amplification reactions using Qiagen&#39;s 2× Hot Start Master Mix (Contains Hot Start Taq DNA Polymerase, and a final concentration of 1.5 mM MgCl 2  and 200 μM of each dNTP). 
     To each reaction, 12.5 μl of Hot Start Master Mix is added to 1 μl of genomic DNA (approximately 200 ng genomic DNA for the mutant DNA sample and the wild-type DNA sample), which is purified from human blood using Pharmacia Amersham Blood purification kits. By another approach, the DNA samples from a plurality of subjects can be mixed prior to generation of the extension products. In this case, approximately 100 ng of wild-type genomic DNA is mixed with approximately 100 ng of mutant N370S genomic DNA. Primers are added to achieve a final concentration of 0.5 μM for each primer and a final volume of 25 μl is obtained by adjusting the volume with ddH 2 O. 
     Thermal cycling is performed using the following parameters: 15 minutes @ 95° C. for 1 cycle; 30 seconds @ 94° C., one minute @ 57° C., and one minute 30 seconds @ 72° C. for 35 cycles; and 10 minutes @ 72° C. for 1 cycle. After amplification, the extension products generated from the wild-type and mutant templates (if un-mixed samples) are separated from the PCR reactants using a PCR Clean Up kit (Qiagen). Then, approximately 10 :L of the wild-type and mutant DNA are removed from each tube and gently mixed in a single reaction vessel. This preparation is then denatured at 95° C. for 1 minute and rapidly cooled to 4° C. for 5 minutes. Finally, the preparation is brought to 65° C. for an additional 1.5 minutes. The extension products generated from the mixed sample (wild-type DNA and mutant DNA mixed prior to amplification) can be stored until loaded onto a DHPLC column. 
     Next, the extension products are loaded on to a 50×4.6 mm ion pair reverse phase HPLC column that is equilibrated in degassed Buffer A (0.1 M triethylamine acetate (TEAA) pH 7.0) at 56° C. A linear gradient of 40%-50% of degassed Buffer B (0.1 M triethylamine acetate (TEAA) pH 7.0 and 25% acetonitrile) is then performed over 2.5 minutes at a flow rate of 0.9 ml/min at 56° C., followed by a linear gradient of 50%-55.3% Buffer B over 0.5 minutes, and finally a linear gradient of 55.3%-61% Buffer B over 4 minutes. U.V. absorption is monitored at 260 nm, recorded and plotted against retention time. 
     When the loaded sample is un-mixed extension products, the extension products generated from only the wild-type or mutant DNA template, it is difficult to distinguish the wild type homoduplex from the N370S mutant homoduplex. When the loaded sample is the mixed extension products, the extension products generated from the mixed DNA templates (wild-type and mutant DNA mixed prior to amplification), or the extension products (generated from wild type and mutant DNA separately) that were mixed after amplification, heteroduplex elution behavior is detected. By practicing the methods taught in this example, the throughput of diagnostic screening can be drastically improved and the cost of identifying genetic traits can be significantly reduced. The example below describes an approach that was used to diagnostically screen patient samples for the presence or absence of polymorphisms or mutations on genes associated with HNPCC. 
     Example 8 
     Sets of primers for PCR amplification were designed for every exon of the MLH1 and MSH2 genes. The primers were designed from sequence information that was available from GenBank or from sequence information obtained from Ambry Genetics Corporation. Information regarding mutations or polymorphisms was obtained from The Human Gene Mutation Database. 
     Primer sets and PCR stacking groups were designed for optimal sensitivity for TTGE, as described above. DNA from one individual was amplified with each primer set in a separate reaction, then stacked in average groups of three fragments/gel for gel analysis. Preferably, one of the primers in each primer set contained a GC-clamp. It was discovered that the addition of a GC-clamp significantly altered the melting profile of the DNA extension product. Further, proper positioning of the GC-clamp served to level the melting profile. It was desired to position the GC-clamp so that a tight single melting domain across the fragment was created. Proper positioning of the GC-clamp was often times needed to prevent the GC-clamp from masking the presence of a mutation or polymorphism (e.g., if the mutation or polymorphism is too close to the GC-clamp). Software was also used to optimize primer design. For example, many primers were designed with the aid of Pruner Premiere 4.0 and 5.0 and appropriate positioning of the GC-clamps was determined using WinMelt software from BioRad. To maintain sensitivity of the test, the primers were designed to anneal at a minimum of 40 base pairs either upstream or downstream of the nearest known mutation in the intronic region of the genes. 
     Optimization was determined for each primer set. Optimization experiments were conducted using Hotstart Master Mix from Qiagen and a Thermocyler from MJ Research. Resulting PCR conditions for all fragments were 15 minutes @ 95° C. for the initial denaturation, then 35 cycles of 30 seconds @ 94° C., 30 seconds @ 46-62° C., and 30 seconds @ 72° C. A final extension was performed at 72° C. for 10 minutes. Approximately 15 ul PCR reactions contained 7.5 ul Qiagen 2× Hotstart Master Mix, 50-200 ng genomic DNA, sense and antisense primer for each fragment at a final concentration of 0.5-1 μM. Prior to gel loading and stacking of gel groups PCR samples were heated and re-annealed to provide best heteroduplex formation. PCR product was heated to 95° C. for 5 min, 50° C. for 10 min, then brought to 4° C. 
     PCR products (approximately 4-8 μl each depending on signal strength) were then assembled for groups of equal melting characteristics and mixed with loading dye consisting of 70% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 2 mM EDTA). DNA was separated on denaturing gels (7 M urea, 8% acrylamide/bis(37.5:1) in 50 mM Tris, 25 mM acetic acid, 1.25 mM EDTA) for 3-5 hours at 125 V or 150 V on the Dcode system. (Biorad). Temperature ranged from 45.5° C. to 64° C. with ramp rates of 1.0-1.5° C./hr depending on gel groups. The gels were stained in 1 :g/ml ethidium bromide in 1.25×TAE for 3 minutes and destained in 1.25×TAE buffer for 20 minutes. The gels were photographed using the Gel Doc 1000 system (BioRad). Table 2 below lists the primers used in this assay. TABLE D shows the TTGE gel grouping (MLH or MSH stacking group) and temperatures used for TTGE separation (under “Run group”). TABLE D also names the extension products generated from the various primer sets employed and the positions of each fragment on the gel after separation (listed in order). Previous experiments, described above, have demonstrated that extension products generated from primers that are any number between 1-75 nucleotides upstream or downstream from the primers listed in TABLE A (e.g., the primer sets listed in Table 2) can be grouped and efficiently separated in accordance with rules set forth herein. Preferably, the primers listed in Table 2 are used to generate extension products that are grouped according to TABLE D and are separated on the basis of melting behavior (e.g., TTGE). In Table 2, the notation “(*)-” indicates the presence of a GC-rich clamp sequence, the sequence of which is given at the bottom of the Table. 
     
       
         
           
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Primer name 
                 SEQ ID 
                 Primer sequence 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-1A-s: 
                 3 
                 5′ (*)-CAATAGCTGCCGCTGA 3′ 
                   
               
               
                 MLH1-1A-as: 
                 4 
                 5′ CGCTGGATAACTTCCC 3′ 
               
               
                   
               
               
                 MLH1-1B-s: 
                 5 
                 5′ GGCGGGGGAAGTTAT 3′ 
               
               
                 MLH1-1B-as: 
                 6 
                 5′ (*)-CGCGCCATTGAGTGAC 3′ 
               
               
                   
               
               
                 MLH1-1C-s: 
                 7 
                 5′ (*)-CAAAGAGATGATTGAGAAC 3′ 
               
               
                 MLH1-1C-as: 
                 8 
                 5′ CATGCCTCTGCCCGG 3′ 
               
               
                   
               
               
                 MLH1-1D-s: 
                 9 
                 5′ (*)-GGAAGAACGTGAGCACGA 3′ 
               
               
                 MLH1-1D-as: 
                 10 
                 5′ CATTAGCTGGCCGCTG 3′ 
               
               
                   
               
               
                 MLH1-2A-s: 
                 16 
                 5′ (*)-TTATCATTGCTTGGCT 3′ 
               
               
                 MLH1-2A-as: 
                 17 
                 5′ TTGTCTTGGATCTGAATC 3′ 
               
               
                   
               
               
                 MLH1-2B-s: 
                 18 
                 5′ (*)-GCAAAATCCACAAGTATT 3′ 
               
               
                 MLH1-2B-as: 
                 19 
                 5′ CCTGACTCTTCCATGAA 3′ 
               
               
                   
               
               
                 MLH1-3A-s: 
                 23 
                 5′ (*)-GGGAATTCAAAGAGAT 3′ 
               
               
                 MLH1-3A-as: 
                 24 
                 5′ TTCTTGAATCTTTAGCTT 3′ 
               
               
                   
               
               
                 MLH1-3B-s: 
                 25 
                 5′ ATATTGTATGTGAAAGGTTCAC 3′ 
               
               
                 MLH1-3B-as: 
                 26 
                 5′ (*)-ACCAAACCTTATTTATCTATGT 3′ 
               
               
                   
               
               
                 MLH1-4A-s4 
                 32 
                 5′ GGTGAGGTGACAGTGGGT 3′ 
               
               
                 MLH1-4A-as4 
                 33 
                 5′ (*)-TGAATATATATGAGTAAAAGAAGTCAG 3′ 
               
               
                   
               
               
                 MLH1-4B-s2 
                 34 
                 5′ TCATGTTACTATTACAACGAAAA 3′ 
               
               
                 MLH1-4B-as2 
                 35 
                 5′ (*)-GATAACACTGGTGTTGAGACA 3′ 
               
               
                   
               
               
                 MLH1-5a-s: 
                 39 
                 5′ (*)-GGGATTAGTATCTATCTCT 3′ 
               
               
                 MLH1-5A-as: 
                 40 
                 5′ GGCTTTCAGTTTTCC 3′ 
               
               
                   
               
               
                 MLH1-5B-s2: 
                 41 
                 5′ CTGAAAGCCCCTCCTA 3′ 
               
               
                 MLH1-5B-as2: 
                 42 
                 5′ (*)-AGCTTCAACAATTTACTCTC 3′ 
               
               
                   
               
               
                 MLH1-5C-s2: 
                 43 
                 5′ CAAGGGACCCAGATCAC 3′ 
               
               
                 MLH1-5C-as2: 
                 44 
                 5′ (*)-CCAATATTTATACAAACAAAGC 3′ 
               
               
                   
               
               
                 MLH1-5D-s 
                 45 
                 5′ (*)-TTTGTTATATTTTCTCATTAGAG 3′ 
               
               
                 MLH1-5D-s 
                 46 
                 5′ ATTCTTACCGTGATCTGG 3′ 
               
               
                   
               
               
                 MLH1-6-5-s 
                 50 
                 5′ (*)-ATTCACTATCTTAAGACCTCGCT 3′ 
               
               
                 MLH1-6-5-as 
                 51 
                 5′ CTAGAACACATTACTTTGATGACAA 3′ 
               
               
                   
               
               
                 MLH1-7-s: 
                 55 
                 5′ TAACTAAAAGGGGGCT 3′ 
               
               
                 MLH1-7-as: 
                 56 
                 5′ (*)-TTTATTGTCTCATGGCT 3′ 
               
               
                   
               
               
                 MLH1-8A-s: 
                 60 
                 5′ (*)-GCTGGTGGAGATAAGG 3′ 
               
               
                 MLH1-8A-as: 
                 61 
                 5′ TGTCCACGGTTGAGG 3′ 
               
               
                   
               
               
                 MLH1-8B-s: 
                 62 
                 5′ GGGGGCAAGGAGAGACAGTAG 3′ 
               
               
                 MLH1-8B-as2: 
                 63 
                 5′ (*)-ATATAGGTTATCGACATACC 3′ 
               
               
                   
               
               
                 MLH1-8C-s2: 
                 64 
                 5′ AAATGCTGTTAGTC 3′ 
               
               
                 MLH1-8C-as: 
                 65 
                 5′ (*)-TCTTGAAAGGTTCCAA 3′ 
               
               
                   
               
               
                 MLH1-9A-3-s 
                 69 
                 5′ (*)-GTAATGTTTGAGTTTTGAGTATTTTC 3′ 
               
               
                 MLH1-9A-3-as 
                 70 
                 5′ CAGAAATTTTTCCATGGTCC 3′ 
               
               
                   
               
               
                 MLH1-9B-s 
                 71 
                 5′ (*)-CAAAGTTAGTTTATGGGAAGGA 3′ 
               
               
                 MLH1-9B-as 
                 72 
                 5′ GAAGAGTAAGAAGATGCACTTCTT 3′ 
               
               
                   
               
               
                 MLH1-9C-s 
                 73 
                 5′ (*)-CTTCAAAATGAATGGTTACATAT 3′ 
               
               
                 MLH1-9C-as 
                 74 
                 5′ ATTCCCTGTGGGTGTTTC 3′ 
               
               
                   
               
               
                 MLH1-10-s: 
                 78 
                 5′ (*)-TGAATGTACACCTGTGAC 3′ 
               
               
                 MLH1-10-as: 
                 79 
                 5′ TAGAACATCTGTTCCTTG 3′ 
               
               
                   
               
               
                 MLH1-11A-s: 
                 83 
                 5′ (*)-TTGACCACTGTGTCATC 3′ 
               
               
                 MLH1-11A-as: 
                 84 
                 5′ GTGCAGGAAGTGAACT 3′ 
               
               
                   
               
               
                 MLH1-11B-s: 
                 85 
                 5′ (*)-CAGAATGTGGATGTTAATG 3′ 
               
               
                 MLH1-11B-as: 
                 86 
                 5′ GGAGGAATTGGAGCC 3′ 
               
               
                   
               
               
                 MLH1-11C-s4: 
                 87 
                 5′ CAGCAGCACATCGAGAG 3′ 
               
               
                 MLH1-11C-as4: 
                 88 
                 5′ (*)-ATCTGGGCTCTCACGTCT 3′ 
               
               
                   
               
               
                 MLH1-12B-s: 
                 92 
                 5′ (*)-TTTTTTTTAATACAGACTTTG 3′ 
               
               
                 MLH1-12B-as: 
                 93 
                 5′ GTGACAATGGCCTGG 3′ 
               
               
                   
               
               
                 MLH1-12C-s: 
                 94 
                 5′ CATTTCTGCAGCCTCT 3′ 
               
               
                 MLH1-12C-as: 
                 95 
                 5′ (*)-TTTTTGGCAGCCACT 3′ 
               
               
                   
               
               
                 MLH1-12D-s3: 
                 96 
                 5′ AGCCCCTGCTGAAGTG 3′ 
               
               
                 MLH1-12D-as3: 
                 97 
                 5′ (*)-AGAAGGCAGTTTTATTACAGA 3′ 
               
               
                   
               
               
                 MLH1-12E-s: 
                 98 
                 5′ (*)-TGTCCAGTCAGCCCCA 3′ 
               
               
                 MLH1-12E-as: 
                 99 
                 5′ CTCTGATTTTTGGCAGC 3′ 
               
               
                   
               
               
                 MLH1-13A-s: 
                 106 
                 5′ (*)-AATTTGGCTAAGTTTAA 3′ 
               
               
                 MLH1-13A-as: 
                 107 
                 5′ GGAATCATCTTCCACC 3′ 
               
               
                   
               
               
                 MLH1-13B-s2: 
                 108 
                 5′ (*)-CATTGCAGAAAGAGACATC 3′ 
               
               
                 MLH1-13B-as3: 
                 109 
                 5′ CGCCCGCCGCGGTGAGGTTAATGATCCTTCT 3′ 
               
               
                   
               
               
                 MLH1-13C-s1: 
                 110 
                 5′ (*)-TGATTCCCGAAAGGAAATGAC 3′ 
               
               
                 MLH1-13C-as1: 
                 111 
                 5′ CAGGCCACAGCGTTTACGTACCCTCATG 3′ 
               
               
                   
               
               
                 MLH1-13D-s: 
                 112 
                 5′ (*)-ATTAACCTCACTAGTGTTTTG 3′ 
               
               
                 MLH1-13D-as: 
                 113 
                 5′ TGAGGCCCTATGCATC 3′ 
               
               
                   
               
               
                 MLH1-14A-s: 
                 117 
                 5′ (*)-GGTCAATGAAGTGGGG 3′ 
               
               
                 MLH1-14A-as: 
                 118 
                 5′ CCACGAAGGAGTGGTTA 3′ 
               
               
                   
               
               
                 MLH1-14B-s: 
                 119 
                 5′ AGTTCTCCGGGAGATG 3′ 
               
               
                 MLH1-14B-as: 
                 120 
                 5′ (*)-TACCTCATGCTGCTCTC 3′ 
               
               
                   
               
               
                 MLH1-15-s: 
                 124 
                 5′ TTCAGGGATTACTTCTC 3′ 
               
               
                 MLH1-15-as: 
                 125 
                 5′ (*)-GAAAAATTTAACATACTACA 3′ 
               
               
                   
               
               
                 MLH1-16A-s: 
                 129 
                 5′ (*)-GCCATTCTGATAGTGGA 3′ 
               
               
                 MLH1-16A-as2: 
                 130 
                 5′ TCTAAGGCAAGCATGGCAA 
               
               
                   
               
               
                 MLH1-16B-s: 
                 131 
                 5′ GCACCGCTCTTTGA 3′ 
               
               
                 MLH1-16B-as: 
                 132 
                 5′ (*)-GTATAAGAATGGCTGTCA 3′ 
               
               
                   
               
               
                 MLH1-16C-s2: 
                 133 
                 5′ GGCTGAGATGCTTGCAG 3′ 
               
               
                 MLH1-16C-as2: 
                 134 
                 5′ (*)-CATGAGCCACCGCAC 3′ 
               
               
                   
               
               
                 MLH1-17-s: 
                 138 
                 5′ (*)-TGTTTAAACTATGACAGCA 3′ 
               
               
                 MLH1-17-as: 
                 139 
                 5′ TGGTCATTTGCCCTT 3′ 
               
               
                   
               
               
                 MLH1-18A-s: 
                 143 
                 5′ (*)-TGTGATCTCCGTTTAGAA 3′ 
               
               
                 MLH1-18A-as2: 
                 144 
                 5′ CTGAGAGGGTCGACTCC 3′ 
               
               
                   
               
               
                 MLH1-18B-s3: 
                 145 
                 5′ (*) TGCGCTATGTTCTATTCCA 3′ 
               
               
                 MLH1-18B-as3: 
                 146 
                 5′ GCCGCCCCCGCCCGCTAGTCCTGGGGTGCCA 3′ 
               
               
                   
               
               
                 MLH1-19A-s: 
                 150 
                 5′ CAAGTCTTTCCAGACCC 3′ 
               
               
                 MLH1-19A-as: 
                 151 
                 5′ (*)-TGTATAGATCAGGCAGGT 3′ 
               
               
                   
               
               
                 MLH1-19B-s4 
                 153 
                 5′ AAGCCTTGCGCTCACAC 3′ 
               
               
                 MLH1-19B-as4 
                 155 
                 5′ (*)-AATAACCATATTTAACACCTCTCAA 3′ 
               
               
                   
               
               
                 MLH1-19C-s: 
                 152 
                 5′ (*)-CAGAAGATGGAAATATCCTGC 3′ 
               
               
                 MLH1-19C-as: 
                 153 
                 5′ CCGCCCGTGTATATCACACTTTGATACAACACT3′ 
               
               
                   
               
               
                 (*) clamp is 
                 344 
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG 
               
               
                   
               
               
                 MSH2-2B-s3 
                 167 
                 5′ (*)-GGAGCAAAGAATCTGCAGAG 3′ 
               
               
                 MSH2-2B-as3 
                 168 
                 5′ TAATTACCTTATATGCCAAATACCA 3′ 
               
               
                   
               
               
                 MSH2-2C-s: 
                 165 
                 5′ ATAAGGCATCCAAGGAGAA 3′ 
               
               
                 MSH2-2C-as: 
                 166 
                 5′ (*)-ATCTACTTAAAATACTAAAACACAAT 3′ 
               
               
                   
               
               
                 MSH2-3A-s: 
                 174 
                 5′ (*)-AACATTTTATTAATAAGGTTC 3′ 
               
               
                 MSH2-3A-as: 
                 175 
                 5′ ATTGCCAGGAGAAGC 3′ 
               
               
                   
               
               
                 MSH2-3B-s2: 
                 176 
                 5′ (*)-ATTTTTACTTAGGCTTCTCCTG 3′ 
               
               
                 MSH2-3B-as2: 
                 177 
                 5′ CAGTTTCCCCATGTCTCC 3′ 
               
               
                   
               
               
                 MSH2-3C-s: 
                 178 
                 5′ AATGTGTTTTACCCGGAG 3′ 
               
               
                 MSH2-3C-as: 
                 179 
                 5′ (*)-CTTAAATGAAACAGTATCATGTC 3′ 
               
               
                   
               
               
                 MSH2-4A-s: 
                 183 
                 5′ (*)-TCCTTTTCTCATAGTAGTTTA 3′ 
               
               
                 MSH2-4A-as: 
                 184 
                 5′ TTGAGGTCCTGATAAATG 3′ 
               
               
                   
               
               
                 MSH2-4A-s2: 
                 185 
                 5 (*)-TTTCTTTCAAAATAGATAATTC 3′ 
               
               
                 MSH2-4A-as2: 
                 186 
                 5′ TTTTTGCCTTTCAACA 3′ 
               
               
                   
               
               
                 MSH2-4B-2s: 
                 187 
                 5′ ATTTATCAGGACCTCAA 3′ 
               
               
                 MSH2-4B-2as: 
                 188 
                 5′ (*)-TGTAATTCACATTTATAATC 3′ 
               
               
                   
               
               
                 MSH2-4C-s: 
                 189 
                 5′ ATTGCCAGAAATGGAG 3′ 
               
               
                 MSH2-4C-as: 
                 190 
                 5′ (*)-ACATATTTACATTATATATATTGT 3 
               
               
                   
               
               
                 MSH2-5A-s: 
                 194 
                 5′ (*)-TTCATTTTGCATTTGTT 3′ 
               
               
                 MSH2-5A-as: 
                 195 
                 5′ CTTGATTACCGCAGAC 3′ 
               
               
                   
               
               
                 MSH2-5B-s: 
                 196 
                 5′ (*)-ATCTTCGATTTTTAAATTC 3′ 
               
               
                 MSH2-5B-as: 
                 197 
                 5′ AAAGGTTAAGGGCTCTG 3′ 
               
               
                   
               
               
                 MSH2-6A-s: 
                 203 
                 5′ (*)-GTTTTTCATGGCGTAG 3′ 
               
               
                 MSH2-6A-as: 
                 204 
                 5′ ACTGAGAGCCAGTGGTA 3′ 
               
               
                   
               
               
                 MSH2-6B-s2: 
                 205 
                 5′ TTTACTAGGGTTCTGTTGAAGA 3′ 
               
               
                 MSH2-6B-as: 
                 206 
                 5′ (*)-ATACCTCTCCTCTATTCTG 3′ 
               
               
                   
               
               
                 MSH2-6C-s: 
                 207 
                 5′ TCAAGGACAAAGACTTGT 3′ 
               
               
                 MSH2-6C-as: 
                 208 
                 5′ (*)-CATATTACAATAAGTGGTATAAT 3′ 
               
               
                   
               
               
                 MSH2-7A-s: 
                 212 
                 5′ (*)-GTTGAGACTTACGTGCTT 3′ 
               
               
                 MSH2-7A-as2: 
                 213 
                 5′ CAATTCTGCATCTTCTACAAA 3′ 
               
               
                   
               
               
                 MSH2-7B-s2: 
                 214 
                 5′ (*)-ATTTCAGATTGAATTTAGTGG 3′ 
               
               
                 MSH2-7B-as2: 
                 215 
                 5′ AGTTTGCTGCTTGTCTTTG 3′ 
               
               
                   
               
               
                 MSH2-7C-s3: 
                 216 
                 5′ GACTTGCCAAGAAGTTT 3′ 
               
               
                 MSH2-7C-as2: 
                 217 
                 5′ (*)-TGAGTCACCACCACCAAC 3′ 
               
               
                   
               
               
                 MSH2-8A-s: 
                 221 
                 5′ (*)-TTTGGATCAAATGATGC 3′ 
               
               
                 MSH2-8A-as: 
                 222 
                 5′ ATCAGTAAGAGGAGTCACA 3′ 
               
               
                   
               
               
                 MSH2-8B-s: 
                 223 
                 5′ TTGTGACTCCTCTTACTG 3′ 
               
               
                 MSH2-8B-as: 
                 224 
                 5′ (*)-AATAACTACTGCTTAAATTAA 3′ 
               
               
                   
               
               
                 MSH2-8C-s: 
                 225 
                 5′ CTGACTTCTCCAAGTTTC 3′ 
               
               
                 MSH2-8C-as: 
                 226 
                 5′ (*)-GTGCTACAATTAGATACTAAA 3′ 
               
               
                   
               
               
                 MSH2-8D-s: 
                 227 
                 5′ AGAAATTATTGTTGGCAGTT 3′ 
               
               
                 MSH2-8D-as: 
                 228 
                 5′ (*)-ATTGCATACCTGATCCATATC 3′ 
               
               
                   
               
               
                 MSH2-9A-s2: 
                 232 
                 5′ (*)-AATATTTGCTTTATAATTTC 3′ 
               
               
                 MSH2-9A-as2: 
                 233 
                 5′ AGAATTATTCCAACCTC 3′ 
               
               
                   
               
               
                 MSH2-10A-s: 
                 237 
                 5′ (*)-GAATTACATTGAAAAATGG 3′ 
               
               
                 MSH2-10A-as: 
                 238 
                 5′ TTAATCTGTTTGCCAGG 3′ 
               
               
                   
               
               
                 MSH2-10B-s2: 
                 239 
                 5′ TCTTCTTGATTATCAAGGC 3′ 
               
               
                 MSH2-10B-as2: 
                 240 
                 5′ (*)-ACACCATTCTTCTGGATA 3′ 
               
               
                   
               
               
                 MSH2-10C-s3: 
                 241 
                 5′ TGCACAGTTTGGATATTACTT 3′ 
               
               
                 MSH2-10C-as3: 
                 242 
                 5′ (*)-GTAAAACTTATCATAGAACATTCAC 3′ 
               
               
                   
               
               
                 MSH2-11A-s2: 
                 246 
                 5′ (*)-TTTGGATATGTTTCACGTA 3′ 
               
               
                 MSH2-11A-as2: 
                 247 
                 5′ CTTTAACAATGGCATCCT 3′ 
               
               
                   
               
               
                 MSH2-11B-s2: 
                 248 
                 5′ (*)-GCAAATTGACTTCTTTAAATG 3′ 
               
               
                 MSH2-11B-as2: 
                 249 
                 5′ ATGGCTTGCGAAAATAAC 3′ 
               
               
                   
               
               
                 MSH2-12A-s 
                 253 
                 5′ (*)-AGGAAATGGGTTTTGAA 3′ 
               
               
                 MSH2-12A-as: 
                 254 
                 5′ GAGCTAACACATCATTGAGT 3′ 
               
               
                   
               
               
                 MSH2-12B-s: 
                 255 
                 5′ (*)-ATTTTTATACAGGCTATGTAG 3′ 
               
               
                 MSH2-12B-as: 
                 256 
                 5′ ACATATGGAACAGGTGCT 3′ 
               
               
                   
               
               
                 MSH2-12C-s: 
                 257 
                 5′ TGGAGCACCTGTTCCAT 3′ 
               
               
                 MSH2-12C-as: 
                 258 
                 5′ (*)-AACAAAACGTTACCCCC 3′ 
               
               
                   
               
               
                 MSH2-12E-s: 
                 259 
                 5′ CAGCTTTGCTCACGTGTCA 3′ 
               
               
                 MSH2-12E-as: 
                 260 
                 5′ (*)-CATCTTGAACTTCAACACAAGC 3′ 
               
               
                   
               
               
                 MSH2-13A-s: 
                 264 
                 5′ (*)-TAGGACTAACAATCCATT 3′ 
               
               
                 MSH2-13A-as: 
                 265 
                 5′ TGGGCCATGAGTACTA 3′ 
               
               
                   
               
               
                 MSH2-13B-s: 
                 266 
                 5′ (*)-ATGGGAGGTAAATCAAC 3′ 
               
               
                 MSH2-13B-as: 
                 267 
                 5′ GACTCCTTTCAATTGACT 3′ 
               
               
                   
               
               
                 MSH2-13C-s4: 
                 268 
                 5′ TTGTGGACTGCATCTTAGCC 3′ 
               
               
                 MSH2-13C-5as: 
                 269 
                 5′ (*)-TCACAGGACAGAGACATACATTTC 3′ 
               
               
                   
               
               
                 MSH2-14A-s3 
                 273 
                 5′ (*)-GTATGTGTATGTTACCACATT 3′ 
               
               
                 MSH2-14A-as3 
                 274 
                 5′ TAGTTAAGGTCTCTTCAGTG 3′ 
               
               
                   
               
               
                 MSH2-14B-s 
                 275 
                 5′ ATAATCTACATGTCACAGCA 3′ 
               
               
                 MSH2-14B-as 
                 276 
                 5′ (*)-GAATAAGGCAATTACTGAT 3′ 
               
               
                   
               
               
                 MSH2-15A-s 
                 280 
                 5′ GTCTCTTCTCATGCTGTC 3′ 
               
               
                 MSH2-15A-as 
                 281 
                 5′ (*)-AATAGAGAAGCTAAGTTAAAC 3′ 
               
               
                   
               
               
                 MSH2-16A-s 
                 285 
                 5′ TTACTAATGGGACATTCACATG 3′ 
               
               
                 MSH2-16A-as 
                 286 
                 5′ (*)-ACAATAGCTTATCAATATTACCTTC 3′ 
               
               
                   
               
               
                 * clamp is 
                 344 
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG 
               
               
                   
               
            
           
         
       
     
     In particular embodiments of the invention, primers used to amplify DNA regions from patient samples are labeled with fluorescent tags. Fluorescently tagged primers are used to detect the presence of PCR products without chemical staining as well as the origins of a product when two or more reaction products are mixed and analyzed in the same gel lane. 
     Example 9 
     In this example, fluorescently labeled primers that detect the presence of absence of polymorphisms in the CTFR gene were employed. Exon 10 of the CFTR gene was amplified with a primer set that detects the entire exon using a PCR protocol similar to that of Example 8. PCR was performed as described in Example 8 with a primer set that was modified with Texas Red (primers were obtained from MWG Biotech), and a second primer set that was modified with Oregon Green (also from MWG). Extension products were analyzed on TTGE side by side after being forced into a heteroduplex against themselves or by mixing with a control DNA. The extension products were analyzed on TTGE and the common mutation for deltaF508 and polymorphism M470V was observed. 
     Results revealed the same banding pattern on TTGE for each individual fragment regardless of the modification state of the primer. Results also indicate the homozygous state of the DNA samples if the samples were mixed with wildtype DNA, which appears as a visually apparent heterozygous banding pattern ( FIG. 7 , Panel A). Poststaining of TTGE gels in EtBr also showed the same banding pattern for those products amplified with Texas Red modified or Oregon Green modified primers and unmodified primers. ( FIG. 7 , Panels B and C). 
     This example demonstrates that the use of fluorescently labeled primers allows one to rapidly identify the presence or absence of polymorphisms in an analyzed gene without staining or autoradiography and to rapidly differentiate the identity of individual extension products that are mixed and segregated on the same lane of a TTGE gel. 
     Example 10 
     In one embodiment of the invention, the techniques described above in Example 8 can be used to screen DNA samples isolated from patient blood samples for mutations associated with HNPCC. In some embodiments of the invention, if a DNA sample generates a positive result in the assay, the existence of one or more mutations associated with HNPCC is confirmed with DNA sequencing of the relevant exons. Table E provides primer pairs to be used for the sequencing of each exon of the MSH2 and MLH1 genes, including first and second choices in some instances. A protocol for PCR-based sequencing reactions using these primers, as well as the primer sequences themselves, are also provided. Using the primers, the primer pairings and the protocol provided, a person with skill in the art is able to sequence any or all of the exons of the MSH2 and MLH1 genes and confirm the existence of HNPCC-related or other mutations in the coding sequences of these genes. 
     Example 11 
     Using a protocol similar to that of Example 8, the HNPCC assay is performed with primers that have been modified with a fluorescent label for visualization on a fluorescent imager. In this Example, the short primer (without the GC clamp sequence) of each primer pair listed in Table 2 is modified by the addition of a fluorescent label such as Texas Red (absorption peak 595 nm, emission peak 615 nm) or Oregon Green (absorption peak 496 nm, emission peak 524 nm) (primers are obtained from MWG Biotech). The GC clamp primer is used in the unmodified form. 
     Primer sets and PCR stacking groups are designed for optimal sensitivity for TTGE, as described in Example 8. In particular embodiments, DNA from one individual is amplified with each primer set in a separate reaction, then stacked in average groups of three fragments/gel for gel analysis. PCR conditions for all fragments are as follows: 15 minutes @ 95° C. for the initial denaturation, then 35 cycles of: 30 seconds @ 94° C., 30 seconds @ 47-58.5° C., and 30 seconds @ 72° C. A final extension is performed at 72° C. for 10 minutes. The approximately 15 ul PCR reactions contain 7.5 ul Qiagen 2× Hotstart Master Mix, 50-200 ng genomic DNA, sense and antisense primers for each fragment at a final concentration of 0.5-1 uM. Prior to gel loading and stacking of gel groups, PCR samples are heated and re-annealed to provide best heteroduplex formation. Each PCR product is heated to 95° C. for 5 min, 50° C. for 10 min, then brought to 4° C. PCR products (approximately 4-8 μl each depending on signal strength) are then assembled into groups of products with equal melting characteristics and mixed with loading dye consisting of 70% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 2 mM EDTA). DNA is separated on denaturing gels (7 M urea, 8% acrylamide/bis(37.5:1) in 50 mM Tris, 25 mM acetic acid, 1.25 mM EDTA) for 3-5 hours at 125 V or 150 V on the Dcode system. (Biorad). Temperature ranges from 45° C. to 67° C. are used with ramp rates of 1.0-1.5° C./hr, depending on gel groups. The gels are imaged on a fluorescent image, and images are captured in the respective channel. Gels can also be photographed using the Versadoc 1000 system (BioRad). 
     Resulting images show extension products in the respective channel, e.g. presenting as a red pattern for Texas Red modified primers, and as a green pattern for Oregon Green modified primers. 
     Moreover, since the labeled extension products fluoresce in different spectra, this method allows for the simultaneous visualization of multiple DNA samples at once. For example, if one sample of primer has been previously amplified with Texas Red modified primers and the another with Oregon Green modified primers. one can multiplex the same extension product from 2 or more different DNA samples at the gel stage of the process. 
     In a specific embodiment, DNA from one individual is amplified with each primer set in separate reactions, using short primers labeled with the Texas Red fluorescent tag. DNA from another individual is amplified with primer sets labeled with the Oregon Green fluorescent tag. Prior to gel loading and stacking of gel groups, Texas Red tagged extension product and Oregon Green tagged extension product are mixed at equal ratios, and re-annealed to provide heteroduplex formation. Mixed PCR products are heated to 95° C. for 5 min, 50° C. for 10 min, then brought to 4° C. 
     The PCR products (approximately 4-8 μl of each depending on signal strength) are then assembled into groups of products with equal melting characteristics and mixed with loading dye. DNA is separated on denaturing gels, and gels are imaged on a fluorescent imager. Images for each gel are captured in both channels, after which they are overlayed for viewing of both colors. Whenever the extension products have identical sequence, the banding pattern appears as yellow on the overlay image. If one extension product is missing, the other extension product will be visible (red or green). Moreover, since all products are forced into a heteroduplex, any one homozygous mutation appear as a heterozygous pattern after having been mixed with wildtype sequence. The heterozygous pattern may present as a distinct pattern of 2 yellow, 1 red and 1 green band, or as a compressed yellow pattern of all 4 bands, depending on the specific melting temperature shift of each duplex. Most importantly, this mandatory heteroduplex formation of every fragment in the assay facilitates homozygous detection. This provides an advantage over conventional TTGE, since the homozygous mutations can be the most difficult to resolve on gel. In addition, the cost for analyzing samples is reduced because each gel is loaded with a multiple number of DNA samples. 
     As noted above, heteroduplexes have one or more mismatched base pairs between the two strands comprising the duplex. Creating heteroduplexes in the TTGE samples permits a greater difference in melting temperatures between PCR products with different sequences than would be seen between homoduplexes differing in sequence by only one or a few bases. Heteroduplex formation assists with the melting temperature (T m ) calculations in various Tm calculating software programs, such as the Bio-Rad Winmelt software. In order to get efficient and sensitive TTGE PCR fragments, it is helpful to have the regions of sensitivity be linear within 0.1° C. Consistent predictions of T m  ranges within that level of specificity are difficult to obtain. By increasing the difference in melting temperature of double stranded PCR products in a sample through the formation of heteroduplexes, the need for precise melting temperature predictions is reduced. 
     Another aspect of the invention involves the importance of analysis consistencies in the laboratory. In TTGE, SSCP, DGGE, or any other denaturing assay, the primary determinant for the detection of an abnormality is the mobility shift of the fragment. Even if the assay works technically, the shift may be so slight that it is only apparent if it is known that there is a mutation on the input DNA. Mobility shifts should be visually significant in order to be detected every single time. By creating multicolor heteroduplex under denaturing conditions, color change is added to the visual criteria whereby the mutation can be detected. This additional visual criteria increases the sensitivity of the assay. 
     Although the invention has been described with reference to embodiments and examples, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. 
     
       
         
           
               
               
             
               
                 TABLE A 
               
               
                   
               
             
            
               
                 hMLH1 
                   
               
               
                 genomic seq. and primers 
               
               
                   
               
            
           
           
               
               
            
               
                 5′ upstream seq. 
                   
               
               
                 Aggtagcgggcagtagccgcttcagggagggacgaagagacccagcaacccacagagttgagaaat 
               
               
                 (SEQ ID NO.: 1) 
               
               
                   
               
               
                 Exon 1 
               
               
                                                               ttgactggca ttcaagctgt 
               
               
                 ccaatcaata gctgccgctg aagggtgggg ctggatggcg taagctacag ctgaaggaag 
               
               
                 aacgtgagca cgaggcactg aggtgattgg ctgaaggcac ttccgttgag catctagacg 
               
               
                 tttccttggctctt ctggcgccaa a ATGTCGTTC GTGGCAGGGG TTATTCGGCG GCTGGACGAG   
               
            
           
           
               
               
               
            
               
                 
                   61 
                 
                 
                   ACAGTGGTGA ACCGCATCGC GGCGGGGGAA GTTATCCAGC GGCCAGCTAA TGCTATCAAA 
                 
                   
               
               
                 
                   121 
                 
                   GAGATGATTG AGAACTG gta cggagggagt cgagccgggc tcacttaagg gctacgactt 
               
               
                 181 
                 aacgggccgc gtcactcaat ggcgcggaca cgcctctttg cccgggcaga ggcatgtaca 
               
               
                 241 
                 gcgcatgccc acaacggcgg aggccgccgg gttccctgac gtgccagtca ggccttctcc 
               
               
                 301 
                 ttttccgcag accgtgtgtt tctttaccgc tctcccccga gaccttttaa gggttgtttg 
               
               
                 361 
                 gagtgtaagt ggaggaatat acgtagtgtt gtcttaatgg taccgttaac taagtaagga 
               
               
                 421 
                 agccacttaa tttaaaatta tgtatgcaga acatgcgaag ttaaaagatg tataaaagct 
               
               
                 481 
                 taagatgggg agaaaaacct tttttcagag ggtactgtgt tactgttttc ttgcttttca 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 2) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-1A-s: 
                 5′ (*)-CAATAGCTGCCGCTGA 3′ 
                 (SEQ ID NO.: 3) 
                   
               
               
                   
               
               
                 MLH1-1A-as: 
                 5′ CGCTGGATAACTTCCC 3′ 
                 (SEQ ID NO.: 4) 
               
               
                   
               
               
                 MLH1-1B-s: 
                 5′ GGCGGGGGAAGTTATC 3′ 
                 (SEQ ID NO.: 5) 
               
               
                   
               
               
                 MLH1-1B-as: 
                 5′ (*)-CGCGCCATTGAGTGAC 3′ 
                 (SEQ ID NO.: 6) 
               
               
                   
               
               
                 MLH1-1C-s: 
                 5′ (*)-CAAAGAGATGATTGAGAAC 
                 (SEQ ID NO.: 7) 
               
               
                   
               
               
                 MLH1-1C-AS: 
                 5′ CATGCCTCTGCCCGG 
                 (SEQ ID NO.: 8) 
               
               
                   
               
               
                 MLH1-1D-S: 
                 5′ (*)-GGAAGAACGTGAGCACGA 
                 (SEQ ID NO.: 9) 
               
               
                   
               
               
                 MLH1-1D-AS: 
                 5′ CATTAGCTGGCCGCTG 
                 (SEQ ID NO.: 10) 
               
               
                   
               
               
                 Sense tag: 
                 TCTGCCTTTTTCTTCCATCGGG 
                 (SEQ ID NO.: 11) 
               
               
                   
               
               
                 Antisensense tag: 
                 TCCCCAACCCCCTAAAGCGA 
                 (SEQ ID NO.: 12) 
               
               
                   
               
               
                 MLH1-1seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGGCTTCAGGGAGGGACGAAGA 
                 (SEQ ID NO.: 13) 
               
               
                   
               
               
                 MLH1-1seq-as: 
                 TCCCCAACCCCCTAAAGCGA TGCGCTGTACATGCCTCTGC 
                 (SEQ ID NO.: 14) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 2 
               
            
           
           
               
               
               
            
               
                 2401 
                 gattctcctg ccttagcctc ctgagtagct gggattacag gcatgcgtca ccatgcctgg 
                   
               
               
                 2461 
                 ctaattttgt atttttagta caaatggggt ttctccatgt tggtcaggct ggtctcaaac 
               
               
                 2521 
                 tcctgacctc aggtgatcca cccgccttgg cctcccaaag tgctgggatt atgggtgtga 
               
               
                 2581 
                 gccattgcgc ctggccagaa aattcattga cttcctaaag atttattaac tttctgcatt 
               
               
                 2641 
                 actttttttt ttcccctcca tcgtaaatat aaaagggaat agtagagaaa atcattcaga 
               
               
                 2701 
                 attttatttt ttagtgacat tatttagtga cattttatta gagtcactta ggaacctgag 
               
               
                 2761 
                 gctgaataaa gttcaggtaa aagtaaaatt agttgagaag agacatctgc caaaagaaat 
               
               
                 2821 
                 ctatttttaa cttcacttgc tgtctttcct agaggaacag aaatagtgct gaatgtccta 
               
               
                 2881 
                 ttagaaatga tggttgctct gcccgtctct tccctctctc tcacacaata tgtaaactca 
               
               
                 2941 
                 tacagtgtat gagcctgtaa gacaaaggaa aaacacgtta atgaggcact attgtttgta 
               
               
                 3001 
                 tttggagttt gttatcattg cttggctcat attaaaatat gtacattaga gtagttgcag 
               
               
                 3061 
                 actgataaat tattttctgt ttgatttgcc ag TTTAGATG CAAAATCCAC AAGTATTCAA   
               
               
                 
                   3121 
                 
                 
                   GTGATTGTTA AAGAGGGAGG CCTGAAGTTG ATTCAGATCC AAGACAATGG CACCGGGATC 
                 
               
               
                 
                   3181 
                 
                   AGG gtaagta aaacctcaaa gtagcaggat gtttgtgcgc ttcatggaag agtcaggacc 
               
               
                 3241 
                 tttctctgtt ctggaaacta ggcttttgca gatgggattt tttcactgaa aaattcaaca 
               
               
                 3301 
                 ccaacaataa atatttattg agtacctatt atttgctggg cactgttcag gggatgtgtc 
               
               
                 3361 
                 agtgaataaa atagattaaa atctattctc ttctgatgct tacattatag tggtgggaga 
               
               
                 3421 
                 caaaatgggt ataataaata ttatattaga tagcattaag tgctgtggag aaaactaaag 
               
               
                 3481 
                 cagggaggaa gataggagtg tgcaagccag aaaggttgca attaaattga gtagttcagg 
               
               
                 3541 
                 aaggcttcaa tatggatgtg atatttgaga gaccggtgga agtcaaggag caagttgtga 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 15) 
                   
               
               
                   
               
               
                 Gels: 
               
            
           
           
               
               
               
               
            
               
                 MLH1-2A-s: 
                 5′ (*)-TTATCATTGCTTGGCT 3′ 
                 (SEQ ID NO.: 16) 
                   
               
               
                   
               
               
                 MLH1-2A-as: 
                 5′ TTGTCTTGGATCTGAATC 3′ 
                 (SEQ ID NO.: 17) 
               
               
                   
               
               
                 MLH1-2B-s: 
                 5′ (*)-GCAAAATCCACAAGTATT 3′ 
                 (SEQ ID NO.: 18) 
               
               
                   
               
               
                 MLH1-2B-as: 
                 5′ CCTGACTCTTCCATGAA 3′ 
                 (SEQ ID NO.: 19) 
               
               
                   
               
               
                 MLH1-2seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGTGCCCGTCTCTTCCCTCTCT 
                 (SEQ ID NO.: 20) 
               
               
                   
               
               
                 MLH1-2seq-as: 
                 TCCCCAACCCCCTAAAGCGACCTGAACAGTGCCCAGCAAA 
                 (SEQ ID NO.: 21) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 3 
               
            
           
           
               
               
               
            
               
                 7081 
                 acctgtaatc ccagccactc tggaggctga gacatgaaaa ttgcttgaac ccgggaggcg 
                   
               
               
                 7141 
                 gaggttgcag tgagctgaga tctcgccact gcacttcagc ctgggtgaca gagcaagact 
               
               
                 7201 
                 ctgtctcaaa ggaggttgca gtgagctgag atctcgccac tgcacttcag cctgggtgac 
               
               
                 7261 
                 agagcaagac tctgtctcaa aaaaaaaaaa aacaaaaacc aagaaaagaa aaaaaaactc 
               
               
                 7321 
                 ttctaagagg attttttttt cctggattaa atcaagaaaa tgggaattca aagagatttg 
               
               
                 7381 
                 gaaaaatgag taacatgatt atttactcat ctttttggta tctaacag AA AGAAGATCTG   
               
               
                 
                   7441 
                 
                 
                   GATATTGTAT GTGAAAGGTT CACTACTAGT AAACTGCAGT CCTTTGAGGA TTTAGCCAGT 
                 
               
               
                 
                   7501 
                 
                   ATTTCTACCT ATGGCTTTCG AGGTGAG gta agctaaagat tcaagaaatg tgtaaaatat 
               
               
                 7561 
                 cctcctgtga tgacattgtc tgtcatttgt tagtatgtat ttctcaacat agataaataa 
               
               
                 7621 
                 ggtttggtac cttttacttg ttaaatgtat gcaaatctga gcaaacttaa tgaactttaa 
               
               
                 7681 
                 ctttcaaaga ctgagaattg ttcataaata aactatttta cctgcagaga cctctgatat 
               
               
                 7741 
                 atgtttcttg atggaagtac ccagtaccac ctatgaagtt ttcttgtcaa aaaatcaaat 
               
               
                 7801 
                 gtgaatctga tcattactta gatctaagta ccaatatatg aaaaatatag gagacaagga 
               
               
                 7861 
                 agcatggtaa atgatactga gattgggaga ctacatggaa aaagacttgt tcccttcaac 
               
               
                 7921 
                 agatagacag cagggaaaaa agaatagaga aaggagtaaa gaacctgtag attaaaagac 
               
               
                 7981 
                 atttaaggga catatgaacc aggtccagtg tatagatctt acctaaatcc tgatggagca 
               
               
                 8041 
                 aactataaaa aaattttttt gagacaaatg tttgaataca ggttgactat ttgatggcat 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 22) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-3-s: 
                 5′ (*)-GGGAATTCAAAGAGAT 3′ 
                 (SEQ ID NO.: 23) 
                   
               
               
                   
               
               
                 MLH1-3-as: 
                 5′ TTCTTGAATCTTTAGCTT 3′ 
                 (SEQ ID NO.: 24) 
               
               
                   
               
               
                 MLH1-3B-s: 
                 5′ ATATTGTATGTGAAAGGTTCAC 3′ 
                 (SEQ ID NO.: 25) 
               
               
                   
               
               
                 MLH1-3B-as: 
                 5′ (*)-ACCAAACCTTATTTATCTATGT 
                 (SEQ ID NO.: 26) 
               
               
                   
               
               
                 MLH1-3seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGCAAGACTCTGTCTCAAAGGAGGTT 
                 (SEQ ID NO.: 27) 
               
               
                   
               
               
                 MLH1-3seq-as: 
                 TCCCCAACCCCCTAAAGCGAGACAATGTCATCACAGGAGGAT 
                 (SEQ ID NO.: 28) 
               
               
                   
               
               
                 MLH1-3seq-s2- 
                 cctggattaaatcaagaaaatggg 
                 (SEQ ID NO.: 29) 
               
               
                 internal 
               
               
                   
               
               
                 MLH1-3seq-as2 
                 TCCCCAACCCCCTAAAGCGACATTAAGTTTGCTCAGATTTGCATA 
                 (SEQ ID NO.: 30) 
               
               
                   
               
            
           
           
               
               
            
               
                 to be used with MLH1-3seq-s for PCR and tagged seq 
                   
               
               
                   
               
               
                 Exon 4 
               
            
           
           
               
               
               
            
               
                 10261 
                 gagatgctgt cacacagacc ccgtcatagc acagttcctg agttacatct ttacatactg 
                   
               
               
                 10321 
                 tagtatcctt cttgtgaaaa aagatacaga ttccaaaggt ctgagaaacc aatcttggtt 
               
               
                 10381 
                 ataaagggga aaaatggtca tgggttttta aaatttgttt tgtcttaatt gcatttcaaa 
               
               
                 10441 
                 tttacatttc taaatgaata attgcttata taaagcagtt ttgattaaca atataaaaca 
               
               
                 10501 
                 ctatctattt ggagtgattc ctttacccat ttctgaaggc aagttttaaa aattactaga 
               
               
                 10561 
                 agacacttca ttgagaatat tattaaacat gcctatagtt ctaccacctc aacacaattg 
               
               
                 10621 
                 cttattaaca cattaatgtt ttggtgtgtt ttggactttt taatatgtat ttttcacttg 
               
               
                 10681 
                 ttctagtaat tatgctacag attgatcatt tctttttcaa catgtcatca aagcaagtga 
               
               
                 10741 
                 gcaaagtgct catcgttgcc acatattaat acaaaatgga agcagcagtt cagataacct 
               
               
                 10801 
                 ttccctttgg tgaggtgaca gtgggtgacc cagcagtgag tttttctttc agtctatttt 
               
               
                 10861 
                 cttttcttcc ttag GCTTTG GCCAGCATAA GCCATGTGGC TCATGTTACT ATTACAACGA   
               
               
                 
                   10921 
                 
                   AAACAGCTGA TGGAAAGTGT GCATACAG gt atagtgctga cttcttttac tcatatatat 
               
               
                 10981 
                 tcattctgaa atgtattttt tgcctaggtc tcagagtaat cctgtctcaa caccagtgtt 
               
               
                 11041 
                 atcttttttg gcagagatct tgagtacgtt ttcttttctc cttattgata aattgataat 
               
               
                 11101 
                 cctcaaggat gattattagg tgatactctt acttcatgga ttcttaaaag atatgattta 
               
               
                 11161 
                 acatattaca agtgcctagc aaggtgtctg ttacacgtag gtattttaag taaatggtag 
               
               
                 11221 
                 ctgctgatgt aatttctgcc cctttgccct tcagttgggg tattgctttg gaccgattag 
               
               
                 11281 
                 agggctgtgg ctgggatgct aaaggttcat gtttccttag ctggctcctg agccaccagc 
               
               
                 11341 
                 tcccaccacc tgtgtatacc tgtgctagtt tgccttccca caagtagctg ctggctatct 
               
               
                 11401 
                 gttatgctgg tacagttttc agaaactgat gaatggcctt tgaacagaac aaaaatgaga 
               
               
                 11461 
                 ttcagaataa caaaattgca cctttgtttt tataagcact ggccattcac tagttgaaga 
               
               
                 11521 
                 ctggtaggaa tacctaattc atgccaaaag aaagataatt tttaaaaatc acacaggttg 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 31) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-4A-s4 
                 GGTGAGGTGACAGTGGGT 
                 (SEQ ID NO.: 32) 
                   
               
               
                   
               
               
                 MLH1-4A-as4 
                 (*)-TGAATATATATGAGTAAAAGAAGTCAG 
                 (SEQ ID NO.: 33) 
               
               
                   
               
               
                 MLH1-4B-s2 
                 TCATGTTACTATTACAACGAAAA 
                 (SEQ ID NO.: 34) 
               
               
                   
               
               
                 MLH1-4B-as2 
                 (*)-GATAACACTGGTGTTGAGACA 
                 (SEQ ID NO.: 35) 
               
               
                   
               
               
                 MLH1-4-seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGCATGTCATCAAAGCAAGTGAGC 
                 (SEQ ID NO.: 36) 
               
               
                   
               
               
                 MLH1-4-seq-as: 
                 TCCCCAACCCCCTAAAGCGATGAGACAGGATTACTCTGAGACCT 
                 (SEQ ID NO.: 37) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 5 
               
            
           
           
               
               
               
            
               
                 12961 
                 catttgctgg aagaacagat agtttttcaa atccaattca aggactgggt atggtggctc 
                   
               
               
                 13021 
                 atgcctgtaa tcccagcact ttgggaggcc gaggcaggcg tatccaggag ttcgagacta 
               
               
                 13081 
                 gcctgaccaa catggtgaaa ctccgtctct actaaaaata caaaattagc caggtgtggt 
               
               
                 13141 
                 ggtgggcacc tgtaatctca gctacttggg aggctgaggc aggagaatcg cttgaacctg 
               
               
                 13201 
                 gtaggcggag gttgtagtga gctgagattg tgccattgct ctccagcctg ggaaacaaga 
               
               
                 13261 
                 gcaaaactcc gtctcaaaaa aaaaaaaaat ccaattcaaa tgattatgga agtagtggag 
               
               
                 13321 
                 aaataaacag gaaaatgata aataattaag ataatatata atatggctat attttaatct 
               
               
                 13381 
                 attgttgata tgattttctc ttttcccctt gggattagta tctatctctc tactggatat 
               
               
                 13441 
                 taatttgtta tattttctca ttagAGCAAG TTACTCAGAT GGAAAACTGA AAGCCCCTCC 
               
               
                 13501 
                 TAAACCATGT GCTGGCAATC AAGGGACCCA GATCACGgta agaatggtac atgggagagt 
               
               
                 13561 
                 aaattgttga agctttgttt gtataaatat tggaataaaa aataaaattg cttctaagtt 
               
               
                 13621 
                 ttcagggtaa taataaaatg aatttgcact agttaatgga ggtcccaaga tatcctctaa 
               
               
                 13681 
                 gcaagataaa tgactattgg cttttgtggc atggcagcct gccacgtcct tgtctttttt 
               
               
                 13741 
                 aagggctagg agattcttta ttgggatggc aaaagtcaat ggcagggtag ttgtcattga 
               
               
                 13801 
                 aagaagatta agcttgaccc cagaaggcat gggttagagc ccagccttgt cactcaatgg 
               
               
                 13861 
                 ttgtatgtcc agaggcaagt cacttaacat cccttaaccc cagttttctc atctgtcaaa 
               
               
                 13921 
                 tgaagcaaag aatacttgcc ctcttgactt aaagggtgtc tgatgagaca tatgactgta 
               
               
                 13981 
                 tcattagctg ggagaaagtc catcgtgctg cctatgtata gtgcctcaag ttggtctctt 
               
               
                 14041 
                 tcccttctat gattacacaa agcactccgc tgtcatgtta tccatcccgc ccctccattc 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 38) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-5A-s: 
                 5′ (*)-GGGATTAGTATCTATCTCT 3′ 
                 (SEQ ID NO.: 39) 
                   
               
               
                   
               
               
                 MLH1-5A-as: 
                 5′ GGCTTTCAGTTTTCC 3′ 
                 (SEQ ID NO.: 40) 
               
               
                   
               
               
                 MLH1-5B-s2: 
                 5′ CTGAAAGCCCCTCCTA 3′ 
                 (SEQ ID NO.: 41) 
               
               
                   
               
               
                 MLH1-5B-as2: 
                 5′ (*)-AGCTTCAACAATTTACTCTC 3′ 
                 (SEQ ID NO.: 42) 
               
               
                   
               
               
                 MLH1-5C-s2: 
                 5′ CAAGGGACCCAGATCAC 3′ 
                 (SEQ ID NO.: 43) 
               
               
                   
               
               
                 MLH1-5C-as2: 
                 5′ (*)-CCAATATTTATACAAACAAAGC 3′ 
                 (SEQ ID NO.: 44) 
               
               
                   
               
               
                 MLH1-5D-s 
                 5′ (*)-TTTGTTATATTTTCTCATTAGAG 
                 (SEQ ID NO.: 45) 
               
               
                   
               
               
                 MLH1-5D-s 
                 5′ ATTCTTACCGTGATCTGG 
                 (SEQ ID NO.: 46) 
               
               
                   
               
               
                 MLH1-5seq-s2: 
                 TCTGCCTTTTTCTTCCATCGGGCCCTTGGGATTAGTATCTATCTCT 
                 (SEQ ID NO.: 47) 
               
               
                   
               
               
                 MLH1-5seq-as: 
                 TCCCCAACCCCCTAAAGCGAGGACCTCCATTAACTAGTGCAA 
                 (SEQ ID NO.: 48) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 6 
               
            
           
           
               
               
               
            
               
                 14761 
                 atgcgtcacc atgcccggct aatttttgta tttttagtag agacagggtt tcaccatgtt 
                   
               
               
                 14821 
                 ggccaggctg gtctcgaact cctgacctca ggtgacccac ccaccttggc ctcccaaagt 
               
               
                 14881 
                 tctgggatta cagacgtgag ccactgcacc cagcctgaaa aatatctttg aatgccatgt 
               
               
                 14941 
                 gatactatac ttgtcagttt acatgtgtgt cccactaaat catgtactct cctgagcagg 
               
               
                 15001 
                 atcatgcttt gtcttcatat tttctgtaca aagcaaagac tctgacacaa agctagcccc 
               
               
                 15061 
                 cagtgcatag ttgagaaatc agtgaatgaa tgtgggaggc aggaaaaatg tcctttaatt 
               
               
                 15121 
                 cttctgttaa tgctgtctta tccctggccc cagtcagtgc ttagaactgt gctgttggta 
               
               
                 15181 
                 aatataattg gattcactat cttaagacct cgcttttgcc aggacatctt gggttttatt 
               
               
                 15241 
                 ttcaagtact tctatgaatt tacaagaaaa atcaatcttc tgttcag GTG GAGGACCTTT   
               
               
                 
                   15301 
                 
                 
                   TTTACAACAT AGCCACGAGG AGAAAAGCTT TAAAAAATCC AAGTGAAGAA TATGGGAAAA 
                 
               
               
                 
                   15361 
                 
                   TTTTGGAAGT TGTTGGCAG g tacagtccaa aatctgggag tgggtctctg agatttgtca 
               
               
                 15421 
                 tcaaagtaat gtgttctagt gctcatacat tgaacagttg ctgagctaga tggtgaaaag 
               
               
                 15481 
                 taaaactagc ttacagatag tttctggtca aggtttagcc accaattttg cagtttctct 
               
               
                 15541 
                 catctcccca ggaaagagca gttggtcttt agatcaatga gagctctttt atggcagaca 
               
               
                 15601 
                 aaacaaagtg actctagcca acttgagcta aaaagaaatt tagtggaagg ctaggagtta 
               
               
                 15661 
                 ccacatgaag tgtgtgcagc tgccccttgg agagaataag aaccagggtg cctctgggac 
               
               
                 15721 
                 ttaacatcat tactgtactc cagttgtttt cattcttttc ctgactttgc tctagagtca 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 49) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-6-5-s 
                 (*)-ATTCACTATCTTAAGACCTCGCT 
                 (SEQ ID NO.: 50) 
                   
               
               
                   
               
               
                 MLH1-6-5-as 
                 CTAGAACACATTACTTTGATGACAA 
                 (SEQ ID NO.: 51) 
               
               
                   
               
               
                 MLH1-6seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGCTGTTAATGCTGTCTTATCCCTGG 
                 (SEQ ID NO.: 52) 
               
               
                   
               
               
                 MLH1-6seq-as: 
                 TCCCCAACCCCCTAAAGCGACCATCTAGCTCAGCAACTGTTCA 
                 (SEQ ID NO.: 53) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 7 
               
            
           
           
               
               
               
            
               
                 17461 
                 aatccttcgg ttcacgagct ctgtagagaa aagagaaata accgccaacc aagaaaagat 
                   
               
               
                 17521 
                 tgggagatac tagaataaga cccaggggca ggaagaagcc agtgagaagg agggcatgtt 
               
               
                 17581 
                 gagagctctg agagagaata aaagcagggg ttgttggagc tagcttctca agatgtcctt 
               
               
                 17641 
                 gaggcaaacc agacctttgg gacactctga aaataaaact gaaagtgaag agattgtggg 
               
               
                 17701 
                 ccgaatgtgg tggctcacgc ctgtaatccc agcactttgg gaggtcgagg cgggtggatc 
               
               
                 17761 
                 acctgagatc aggagttcga taccagcctg gccaacatgg cgaaacgcca tctctactaa 
               
               
                 17821 
                 aaatacaaaa aaaattagct gggcctggtg gcaggcgcct ataatcccag ctactcggga 
               
               
                 17881 
                 ggctgaggcg ggagaatcgc ttgagtccag gaggcggagg ttgcagtgag ctgagatcgt 
               
               
                 17941 
                 gccattgcac tccagcctgg gcaacaagag caaaactctg tctcaaaaat aaataaaaat 
               
               
                 18001 
                 aaataaaaaa gagatagtgg cgtgatatcc ttgattctat cagcaaccta taaaagtaga 
               
               
                 18061 
                 gaggagtctg tgttttgatt cagtcacctt tagcattttt atttccatga agtttctgct 
               
               
                 18121 
                 ggtttatttt tctgtgggta aaatattaat aggctgtatg gagatatttt tctttatatg 
               
               
                 18181 
                 tacctttgtt tagattactc aactccacta atttatttaa ctaaaagggg gctctgacat 
               
               
                 18241 
                 ctagtgtgtg tttttggcaa ctcttttctt actcttttgt ttttcttttc cag GTATTCA   
               
               
                 
                   18301 
                 
                   GTACACAATG CAGGCATTAG TTTCTCAGTT AAAAAA gtaa gttcttggtt tatgggggat 
               
               
                 18361 
                 ggttttgttt tatgaaaaga aaaaagggga tttttaatag tttgctggtg gagataaggt 
               
               
                 18421 
                 tatgatgttt cagtctcagc catgagacaa taaatccttg tgtcttctgc tgtttgttta 
               
               
                 18481 
                 tcagcaagga gagacagtag ctgatgttag gacactaccc aatgcctcaa ccgtggacaa 
               
               
                 18541 
                 tattcgctcc atctttggaa atgctgttag tcggtatgtc gataacctat ataaaaaaat 
               
               
                 18601 
                 cttttacatt tattatcttg gtttatcatt ccatcacatt attttggaac ctttcaagat 
               
               
                 18661 
                 attatgtgtg ttaagagttt gctttagtca aatacacagg cttgttttat gcttcagatt 
               
               
                 18721 
                 tgttaatgga gttcttattt cacgtaatca acactttcta ggtgtatgta atctcctaga 
               
               
                 18781 
                 ttctgtggcg tgaatcatgt gttctttcaa ggtcttagtc ttgaaaatat ttatagtgta 
               
               
                 18841 
                 gtagaactat tttatcctcc aatgctcctt cttttccttg tatttccatt atcatcactt 
               
               
                 18901 
                 taggatttca cttatttatc attcaacatt tattaattgc ctctcatatt ccaggctttg 
               
               
                 18961 
                 tgctagaagt tagggatata aagacaaata agatatttcc tgcccttaaa gactagattc 
               
               
                 19021 
                 gtgttgctaa gtcttcatta tcaagaaaag cataagtggg gaaaagtgct tgcattatgg 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 54) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-7-s: 
                 5′ TAACTAAAAGGGGGCT 3′ 
                 (SEQ ID NO.: 55) 
                   
               
               
                   
               
               
                 MLH1-7-as: 
                 5′ (*)-TTTATTGTCTCATGGCT 3′ 
                 (SEQ ID NO.: 56) 
               
               
                   
               
               
                 MLH1-7seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGTTCCATGAAGTTTCTGCTGG 
                 (SEQ ID NO.: 57) 
               
               
                   
               
               
                 MLH1-7seq-as: 
                 TCCCCAACCCCCTAAAGCGACCTTATCTCCACCAGCAAACTA 
                 (SEQ ID NO.: 58) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 8 
               
            
           
           
               
               
               
            
               
                 18001 
                 aaataaaaaa gagatagtgg cgtgatatcc ttgattctat cagcaaccta taaaagtaga 
                   
               
               
                 18061 
                 gaggagtctg tgttttgatt cagtcacctt tagcattttt atttccatga agtttctgct 
               
               
                 18121 
                 ggtttatttt tctgtgggta aaatattaat aggctgtatg gagatatttt tctttatatg 
               
               
                 18181 
                 tacctttgtt tagattactc aactccacta atttatttaa ctaaaagggg gctctgacat 
               
               
                 18241 
                 ctagtgtgtg tttttggcaa ctcttttctt actcttttgt ttttcttttc caggtattca 
               
               
                 18301 
                 gtacacaatg caggcattag tttctcagtt aaaaaagtaa gttcttggtt tatgggggat 
               
               
                 18361 
                 ggttttgttt tatgaaaaga aaaaagggga tttttaatag tttgctggtg gagataaggt 
               
               
                 18421 
                 tatgatgttt cagtctcagc catgagacaa taaatccttg tgtcttctgc tgtttgttta 
               
               
                 18481 
                 tcag CAAGGA GAGACAGTAG CTGATGTTAG GACACTACCC AATGCCTCAA CCGTGGACAA   
               
               
                 
                   18541 
                 
                   TATTCGCTCC ATCTTTGGAA ATGCTGTTAG TCG gtatgtc gataacctat ataaaaaaat 
               
               
                 18601 
                 cttttacatt tattatcttg gtttatcatt ccatcacatt attttggaac ctttcaagat 
               
               
                 18661 
                 attatgtgtg ttaagagttt gctttagtca aatacacagg cttgttttat gcttcagatt 
               
               
                 18721 
                 tgttaatgga gttcttattt cacgtaatca acactttcta ggtgtatgta atctcctaga 
               
               
                 18781 
                 ttctgtggcg tgaatcatgt gttctttcaa ggtcttagtc ttgaaaatat ttatagtgta 
               
               
                 18841 
                 gtagaactat tttatcctcc aatgctcctt cttttccttg tatttccatt atcatcactt 
               
               
                 18901 
                 taggatttca cttatttatc attcaacatt tattaattgc ctctcatatt ccaggctttg 
               
               
                 18961 
                 tgctagaagt tagggatata aagacaaata agatatttcc tgcccttaaa gactagattc 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 59) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-8A-s: 
                 5′ (*)-GCTGGTGGAGATAAGG 3′ 
                 (SEQ ID NO.: 60) 
                   
               
               
                   
               
               
                 MLH1-8A-as: 
                 5′ TGTCCACGGTTGAGG 3′ 
                 (SEQ ID NO.: 61) 
               
               
                   
               
               
                 MLH1-8B-s: 
                 5′ GGGGGCAAGGAGAGACAGTAG 3′ 
                 (SEQ ID NO.: 62) 
               
               
                   
               
               
                 MLH1-8B-as2: 
                 5′ (*)-ATATAGGTTATCGACATACC 3′ 
                 (SEQ ID NO.: 63) 
               
               
                   
               
               
                 MLH1-8C-s2: 
                 5′ AAATGCTGTTAGTC 3′ 
                 (SEQ ID NO.: 64) 
               
               
                   
               
               
                 MLH1-8C-as: 
                 5′ (*)-TCTTGAAAGGTTCCAA 3′ 
                 (SEQ ID NO.: 65) 
               
               
                   
               
               
                 MLH1-8seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGGGTTTATGGGGGATGGTTTTG 
                 (SEQ ID NO.: 66) 
               
               
                   
               
               
                 MLH1-8seq-as: 
                 TCCCCAACCCCCTAAAGCGACGCCACAGAATCTAGGAGATTACA 
                 (SEQ ID NO.: 67) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 9 
               
            
           
           
               
               
               
            
               
                 20401 
                 tattaacctt ccctccccag taaacactcc tgggaacaac acacattgta gaaccacgtt 
                   
               
               
                 20461 
                 gtggtgctgt tcagtatagc aagtaattca gcagagataa gttcttggaa tctcatcttt 
               
               
                 20521 
                 gggatttagt tactaagata cattcaagtt tgagcaaaat aaggtctcag agcttggatt 
               
               
                 20581 
                 cattgttctg ttccagcaat tagagcagta cctggcacat agcacaagtg cttgaaaaca 
               
               
                 20641 
                 ctgactgagt agggtaggtg ggtgagtggg tgggtgggtg ggtgggtgga tggatggatg 
               
               
                 20701 
                 ggaggatggg tgggtgaatg ggtgaacaga caaatggatg gatgaatgga caggcacagg 
               
               
                 20761 
                 aggacctcaa atggaccaag tcttcggggc cctcatttca caaagttagt ttatgggaag 
               
               
                 20821 
                 gaaccttgtg tttttaaatt ctgattcttt tgtaatgttt gagttttgag tattttcaaa 
               
               
                 20881 
                 agcttcagaa tctcttttct aatag AGAAC TGATAGAAAT TGGATGTGAG GATAAAACCC   
               
               
                 
                   20941 
                 
                 
                   TAGCCTTCAA AATGAATGGT TACATATCCA ATGCAAACTA CTCAGTGAAG AAGTGCATCT 
                 
               
               
                 
                   21001 
                 
                   TCTTACTCTT CATCAACC gt aagttaaaaa gaaccacatg ggaaatccac tcacaggaaa 
               
               
                 21061 
                 cacccacagg gaattttatg ggaccatgga aaaatttctg atccataggt ttgattaaac 
               
               
                 21121 
                 atggagaaac ctcatggcaa agtttggttt tattgggaag catgtataat ttttgtccta 
               
               
                 21181 
                 agtctgtgct cagccctccc acatgtgctc attgctggtt gactgttgga gtctggttct 
               
               
                 21241 
                 tacctctaag aggaagccca ggagagggca taaagccagc acactgtcct cacctgatgg 
               
               
                 21301 
                 tgtcagagtc cttacgagta agccctagcc agaacattgc tggaagagat caagggccac 
               
               
                 21361 
                 tgtttgaaat tgcacagcag gatacggaaa aggggtacct taggtatagg cattgtcatt 
               
               
                 21421 
                 aaagaaattg ctaagatact tgagattttc ctgtttaagg aatgagcttt atgatacaaa 
               
               
                 21481 
                 gagcagttct aaaaattagg gagggaatta actaaattaa ttaggatatt tctcaaattc 
               
               
                 21541 
                 ctttacagtt tttgtctctc tgctgatata gtgtttacat gattgttatt tactaaacaa 
               
               
                 21601 
                 atgctatttt gtattgtgct ccttataact taattgttta ttacaaggtt ttgatggtga 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 68) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-9A-3-s 
                 (*)-GTAATGTTTGAGTTTTGAGTATTTTC 
                 (SEQ ID NO.: 69) 
                   
               
               
                   
               
               
                 MLH1-9A-3-as 
                 CAGAAATTTTTCCATGGTCC 
                 (SEQ ID NO.: 70) 
               
               
                   
               
               
                 MLH1-9B-s 
                 (*)-CAAAGTTAGTTTATGGGAAGGA 
                 (SEQ ID NO.: 71) 
               
               
                   
               
               
                 MLH1-9B-as 
                 GAAGAGTAAGAAGATGCACTTCTT 
                 (SEQ ID NO.: 72) 
               
               
                   
               
               
                 MLH1-9C-s 
                 (*)-CTTCAAAATGAATGGTTACATAT 
                 (SEQ ID NO.: 73) 
               
               
                   
               
               
                 MLH1-9C-as 
                 ATTCCCTGTGGGTGTTTC 
                 (SEQ ID NO.: 74) 
               
               
                   
               
               
                 MLH1-9seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGGGTGGGTGAATGGGTGAACA 
                 (SEQ ID NO.: 75) 
               
               
                   
               
               
                 MLH1-9seq-as: 
                 TCCCCAACCCCCTAAAGCGATTTGCCATGAGGTTTCTCCA 
                 (SEQ ID NO.: 76) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 10 
               
            
           
           
               
               
               
            
               
                 23461 
                 tgtctacacc ttaagccgcg gctcccgaag cacctagaac cggaagagtt ggctcactat 
                   
               
               
                 23521 
                 ttagcacaca cacacgtcta taatagtgct ggccacttgg ggttggaatt agtttattta 
               
               
                 23581 
                 tcagcatgtt gtctcccagc acttggtgtg tgtgatatgc agtatgtatt tgcagaatga 
               
               
                 23641 
                 aaagtctgag ggctgacatc atatttccca ctgtgcccag aaagagcaca gttagtccac 
               
               
                 23701 
                 atgagctaat gggggcaaag ggaagtgagg agggagaatg tactgcctta tcatgttttc 
               
               
                 23761 
                 tattacttgg ctgaagtaaa acagtcccaa gccgatagta agatagtggg ctggaaagtg 
               
               
                 23821 
                 gcgacaggta aaggtgcacc tttcttcctg gggatgtgat gtgcatatca ctacagaaat 
               
               
                 23881 
                 gtctttcctg aggtgatttc atgactttgt gtgaatgtac acctgtgacc tcacccctca 
               
               
                 23941 
                 ggacagtttt gaactggttg ctttcttttt attgtttag A TCGTCTGGTA GAATCAACTT   
               
               
                 
                   24001 
                 
                 
                   CCTTGAGAAA AGCCATAGAA ACAGTGTATG CAGCCTATTT GCCCAAAAAC ACACACCCAT 
                 
               
               
                 
                   24061 
                 
                   TCCTGTACCT CAG gtaatgt agcaccaaac tcctcaacca agactcacaa ggaacagatg 
               
               
                 24121 
                 ttctatcagg ctctcctctt tgaaagagat gagcatgcta atagtacaat cagagtgaat 
               
               
                 24181 
                 cccatacacc actggcaaaa ggatgttctg tcccttctta caggtacaag gcacagtttt 
               
               
                 24241 
                 ccttcattta ttcactaatt tagcagaacc tcactaagag cctcctatat gccaggctct 
               
               
                 24301 
                 gcgttagcaa taaaaggaat gccatgcctc accccatcag gaggtgctga tagcttgtag 
               
               
                 24361 
                 gcggagtgga aacagatgtg ctctagaggc tctaaatatt acttctgctg gggtcagttg 
               
               
                 24421 
                 ggaagccaca acagctactg ttcatcttcc ataaaagaca atcagccggg cacagtggct 
               
               
                 24481 
                 cacacctgta aatcccagca ctttgggagg ctgaggtggg tggatcacaa ggtcaggtgt 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 77) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-10-s: 
                 5′ (*)-TGAATGTACACCTGTGAC 3′ 
                 (SEQ ID NO.: 78) 
                   
               
               
                   
               
               
                 MLH1-10-as: 
                 5′ TAGAACATCTGTTCCTTG 3′ 
                 (SEQ ID NO.: 79) 
               
               
                   
               
               
                 MLH1-10seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGGCTGGAAAGTGGCGACAGG 
                 (SEQ ID NO.: 80) 
               
               
                   
               
               
                 MLH1-10seq-s 
                 TCCCCAACCCCCTAAAGCGAGCCAGTGGTGTATGGGATTCA 
                 (SEQ ID NO.: 81) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 11 
               
            
           
           
               
               
               
            
               
                 26221 
                 gatggagtct tgctctgtcg ccaagctgga gtgcagtggc acgatctcgg cttactgcaa 
                   
               
               
                 26281 
                 cctctgactc cctggttgaa gggattctcc tccctcagcc tcccgagtac ctgggattac 
               
               
                 26341 
                 aggcatgcgc caccacgccc agctaatttt tgtattttta gtagagacgt ggtttcatca 
               
               
                 26401 
                 tgttggccag gatggtctcg atctcctgac cttgtgatcc acccgcctcg gcctccccaa 
               
               
                 26461 
                 atgctgggat tacaggcgtg agccaccacg cccggccact tggcatgaat ttaattcccg 
               
               
                 26521 
                 ccataaacct gtgagatagg taattctgtt atatccactt tacaaatgaa gagactgagg 
               
               
                 26581 
                 caaagaaaga tgatgtaact tacgcaaagc tacacagctc ttaagtagca gtgccaatat 
               
               
                 26641 
                 ttgaacacac tcagactcga tcctgaggtt ttgaccactg tgtcatctgg cctcaaatct 
               
               
                 26701 
                 tctggccacc acatacacca tatgtgggct ttttctcccc ctcccactat ctaaggtaat 
               
               
                 26761 
                 tgttctctct tattttcctg acag TTTAGA AATCAGTCCC CAGAATGTGG ATGTTAATGT   
               
               
                 
                   26821 
                 
                 
                   GCACCCCACA AAGCATGAAG TTCACTTCCT GCACGAGGAG AGCATCCTGG AGCGGGTGCA 
                 
               
               
                 
                   26881 
                 
                   GCAGCACATC GAGAGCAAGC TCCTGGGCTC CAATTCCTCC AGGATGTACT TCACCCAG gt 
               
               
                 26941 
                 cagggcgctt ctcatccagc tacttctctg gggcctttga aatgtgcccg gccagacgtg 
               
               
                 27001 
                 agagcccaga tttttgcctg ttatttagga actttctttg caagtattac ctggatagtt 
               
               
                 27061 
                 ttaacatttt cttctttgaa cctagttata aaggtattgt gctgttgttc ctaggcttag 
               
               
                 27121 
                 agtcataagg cctgagctca cttcctcact ttgcctccat ctggaacctt agaccaactt 
               
               
                 27181 
                 cctaggaaaa cgagctgtct gaaaacagaa tagggtgcct cttcaatgtg ctcttcactg 
               
               
                 27241 
                 gagatgttca ggaggaggct actcccacct acacagggtg cagtggaggg tctgggcccc 
               
               
                 27301 
                 agggaggcag caggaagagt ggaaagagcg gaggctctac tgttggacag acctgggtta 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 82) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-11A-s: 
                 5′ (*)-TTGACCACTGTGTCATC 3′ 
                 (SEQ ID NO.: 83) 
                   
               
               
                   
               
               
                 MLH1-11A-as: 
                 5′ GTGCAGGAAGTGAACT 3′ 
                 (SEQ ID NO.: 84) 
               
               
                   
               
               
                 MLH1-11B-s: 
                 5′ (*)-CAGAATGTGGATGTTAATG 3′ 
                 (SEQ ID NO.: 85) 
               
               
                   
               
               
                 MLH1-11B-as: 
                 5′ GGAGGAATTGGAGCC 3′ 
                 (SEQ ID NO.: 86) 
               
               
                   
               
               
                 MLH1-11C-s4: 
                 5′ CAGCAGCACATCGAGAG 3′ 
                 (SEQ ID NO.: 87) 
               
               
                   
               
               
                 MLH1-11C-as4: 
                 5′ CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGATCTGG 
                 (SEQ ID NO.: 88) 
               
               
                   
                 GCTCTCACGTCT 3′ 
               
               
                   
               
               
                 MLH1-11seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGAGACTGAGGCAAAGAAAGATG 
                 (SEQ ID NO.: 89) 
               
               
                   
               
               
                 MLH1-11seq-as: 
                 TCCCCAACCCCCTAAAGCGAAGGCAAAAATCTGGGCTCT 
                 (SEQ ID NO.: 90) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 12 
               
            
           
           
               
               
               
            
               
                 31681 
                 aagatgaaaa agttctagag atagctggtg gtgatggttg cgcaacaatg taaatgccac 
                   
               
               
                 31741 
                 tgagctctca tttaaaaatg gttaaaatgg taaattttat atatatttta ccacaataaa 
               
               
                 31801 
                 aaaaagtctt cttctgggag caccccccca agacaaaaat atgaaaattt tacactgata 
               
               
                 31861 
                 cttccatttc aagataattt taagattata aggattttgc ttaattcttg aattttatac 
               
               
                 31921 
                 ctgtaaacct tttatacttc aaatttcggg cagaattgct tctataacaa tgataattat 
               
               
                 31981 
                 acctcatact agcttctttc ttagtactgc tccatttggg gacctgtata tctatacttc 
               
               
                 32041 
                 ttattctgag tctctccact atatatatat atatatatat atattttttt tttttttttt 
               
               
                 32101 
                 ttttaataca  gACTTTGCTA CCAGGACTTG CTGGCCCCTC TGGGGAGATG GTTAAATCCA   
               
               
                 
                   32161 
                 
                 
                   CAACAAGTCT GACCTCGTCT TCTACTTCTG GAAGTAGTGA TAAGGTCTAT GCCCACCAGA 
                 
               
               
                 
                   32221 
                 
                 
                   TGGTTCGTAC AGATTCCCGG GAACAGAAGC TTGATGCATT TCTGCAGCCT CTGAGCAAAC 
                 
               
               
                 
                   32281 
                 
                 
                   CCCTGTCCAG TCAGCCCCAG GCCATTGTCA CAGAGGATAA GACAGATATT TCTAGTGGCA 
                 
               
               
                 
                   32341 
                 
                 
                   GGGCTAGGCA GCAAGATGAG GAGATGCTTG AACTCCCAGC CCCTGCTGAA GTGGCTGCCA 
                 
               
               
                 
                   32401 
                 
                 
                   AAAATCAGAG CTTGGAGGGG GATACAACAA AGGGGACTTC AGAAATGTCA GAGAAGAGAG 
                 
               
               
                 
                   32461 
                 
                   GACCTACTTC CAGCAACCCC AG gtatggcc ttttgggaaa agtacagcct acctccttta 
               
               
                 32521 
                 ttctgtaata aaactgcctt ctaactttgg cttttcatga atcacttgca tcttctctct 
               
               
                 32581 
                 gcctgacttg ccctctggaa tggtgctgga atggtcctgt ggccttgtcc actgtctgcc 
               
               
                 32641 
                 tttgaccata acttgaaagt cacccaccat agtgtccttt gaaataactt aaatgtccac 
               
               
                 32701 
                 agttccaagc atgagttaaa aacacttcag aatgtagagt agttgttcaa ttgaataaac 
               
               
                 32761 
                 acacacacca gaaaaaaaag caagtttatc ttttattttt agtaaagaat tttgatagag 
               
               
                 32821 
                 cctcaacacc agaaatggct agagagagaa gcctaacata tctggaggat tatttttcat 
               
               
                 32881 
                 cctacttaaa gctgctttca cttttttcag gaaaaaacac acgttctgaa tctaatttat 
               
               
                 32941 
                 aaaactccct ggccgggtgc tgtggctcac acctataatc ccagcacttt gggaggctga 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 91) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-12B-s: 
                 5′ (*)-TTTTTTTTAATACAGACTTTG 3′ 
                 (SEQ ID NO.: 92) 
                   
               
               
                   
               
               
                 MLH1-12B-as: 
                 5′ GTGACAATGGCCTGG 3′ 
                 (SEQ ID NO.: 93) 
               
               
                   
               
               
                 MLH1-12C-s: 
                 5′ CATTTCTGCAGCCTCT 3′ 
                 (SEQ ID NO.: 94) 
               
               
                   
               
               
                 MLH1-12C-as: 
                 5′ (*)-TTTTTGGCAGCCACT 3′ 
                 (SEQ ID NO.: 95) 
               
               
                   
               
               
                 MLH1-12D-s3: 
                 5′ AGCCCCTGCTGAAGTG 3′ 
                 (SEQ ID NO.: 96) 
               
               
                   
               
               
                 MLH1-12D-as3: 
                 5′ (*)-AGAAGGCAGTTTTATTACAGA 3′ 
                 (SEQ ID NO.: 97) 
               
               
                   
               
               
                 MLH1-12E-s: 
                 5′ (*)-TGTCCAGTCAGCCCCA 
                 (SEQ ID NO.: 98) 
               
               
                   
               
               
                 MLH1-12E-as: 
                 5′ CTCTGATTTTTGGCAGC 
                 (SEQ ID NO.: 99) 
               
               
                   
               
               
                 MLH1-12seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGTTTCGGGCAGAATTGCTTC 
                 (SEQ ID NO.: 100) 
               
               
                   
               
               
                 MLH1-12seq-as: 
                 TCCCCAACCCCCTAAAGCGAGCAGAGAGAAGATGCAAGTGATT 
                 (SEQ ID NO.: 101) 
               
               
                 631 bp 
               
               
                   
               
               
                 MLH1-12seq-s2 
                 CAGACTTTGCTACCAGGACTTGCT 
                 (SEQ ID NO.: 102) 
               
               
                 internal 
               
            
           
           
               
               
            
               
                 to be used after amplification with first primer set, but use this for 
                   
               
               
                 seq instead of MLH1-12seq-s 
               
               
                   
               
               
                 ALTERNATIVE FCTL SEQ PRIMER SET: 
               
            
           
           
               
               
               
               
            
               
                 MLH1-12seq-s2 
                 TCTGCCTTTTTCTTCCATCGGGATAGCTGGTGGTGATGGTTGCG 
                 (SEQ ID NO.: 103) 
                   
               
               
                   
               
               
                 MLH1-12seq-as2 
                 TCCCCAACCCCCTAAAGCGACCATTCCAGCACCATTCCAGAG 
                 (SEQ ID NO.: 104) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 13 
               
            
           
           
               
               
               
            
               
                 34801 
                 gcctggaaga catagtgaga ctctctctca aaaaaaaaaa aaaaaaaaaa ggaagtaagc 
                   
               
               
                 34861 
                 attgtgaggg caggtacctt ctctgttttg ttcattgctg gatgtagtta gtatacagca 
               
               
                 34921 
                 gtatctgatg gatggataga tggaggaatg aatgaatgag acttcacaaa ttcagctcac 
               
               
                 34981 
                 ttgctcaagg ccctgcagct ctacgggatg aagctatact ccagagtcct gctacattgg 
               
               
                 35041 
                 ctgtgtggcc agctgctggg atctgagggt tgtcagataa gcagtctacc agagaacaga 
               
               
                 35101 
                 ctgatcttgt tggccttctg ccagcacagg ggttcattca cagctctgta gaaccagcac 
               
               
                 35161 
                 agagaagttg cttgctcctc caaaatgcaa cccacaaaat ttggctaagt ttaaaaacaa 
               
               
                 35221 
                 gaataataat gatctgcact tccttttctt cattgcag AA AGAGACATCG GGAAGATTCT   
               
               
                 
                   35281 
                 
                 
                   GATGTGGAAA TGGTGGAAGA TGATTCCCGA AAGGAAATGA CTGCAGCTTG TACCCCCCGG 
                 
               
               
                 
                   35341 
                 
                 
                   AGAAGGATCA TTAACCTCAC TAGTGTTTTG AGTCTCCAGG AAGAAATTAA TGAGCAGGGA 
                 
               
               
                 
                   35401 
                 
                   CATGAGG gta cgtaaacgct gtggcctgcc tgggatgcat agggcctcaa ctgccaaggt 
               
               
                 35461 
                 tttggaaatg gagaaagcag tcatgttgtc agagtggcca ctacagtttt gctgggcaag 
               
               
                 35521 
                 ctcctcttcc tttactaacc cacaatagca tcagcttaaa gacaattttt gattgggaga 
               
               
                 35581 
                 aaagggagaa aaataatctc tgtttatttt aattagcatt aattggtatt cttgttaaac 
               
               
                 35641 
                 cataggagtc agagtaaatc agccatttca ccaattttca gtttgtttct gtcttagcta 
               
               
                 35701 
                 acagcagtgt aatggtcagc aaaattctta tcttgtgtac tgaatggcat gtcctgttgc 
               
               
                 35761 
                 tgaaagtgca caggcttggg aggtagccat gagctcaaat cctggcacta ccacctctct 
               
               
                 35821 
                 tgtgtgacct tagactcctg acctttctat gcctcagttc tttcttacct ataaaatgaa 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 105) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-13A-s: 
                 5′ (*)-AATTTGGCTAAGTTTAA 3′ 
                 (SEQ ID NO.: 106) 
                   
               
               
                   
               
               
                 MLH1-13A-as: 
                 5′ GGAATCATCTTCCACC 3′ 
                 (SEQ ID NO.: 107) 
               
               
                   
               
               
                 MLH1-13B-s2: 
                 5′ (*)-CATTGCAGAAAGAGACATC 3′ 
                 (SEQ ID NO.: 108) 
               
               
                   
               
               
                 MLH1-13B-as3: 
                 5′ GTGAGGTTAATGATCCTTCT 3′ 
                 (SEQ ID NO.: 109) 
               
               
                   
               
               
                 MLH1-13C-s1: 
                 5′ (*)-TGATTCCCGAAAGGAAATGAC 3′ 
                 (SEQ ID NO.: 110) 
               
               
                   
               
               
                 MLH1-13C-as1: 
                 5′ CAGGCCACAGCGTTTACGTACCCTCATG 3′ 
                 (SEQ ID NO.: 111) 
               
               
                   
               
               
                 MLH1-13D-s: 
                 5′ (*)-ATTAACCTCACTAGTGTTTTG 
                 (SEQ ID NO.: 112) 
               
               
                   
               
               
                 MLH1-13D-as: 
                 5′ TGAGGCCCTATGCATC 
                 (SEQ ID NO.: 113) 
               
               
                   
               
               
                 MLH1-13seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGACTGATCTTGTTGGCCTTCTG 
                 (SEQ ID NO.: 114) 
               
               
                   
               
               
                 MLH1-13seq-as: 
                 TCCCCAACCCCCTAAAGCGATGGCCACTCTGACAACATGA 
                 (SEQ ID NO.: 115) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 14 
               
            
           
           
               
               
               
            
               
                 46261 
                 tggtctccta ttagactctc catttcaaac cattccatga ttttgtcctc cttttgccac 
                   
               
               
                 46321 
                 cttccgagcc tgtaaaaact aatgtttgtg attcctgagg tttctctaat gtcttttaat 
               
               
                 46381 
                 aaagttgacc tcagagatct cgttacctct ctgagttcct gctttgtctt agattttgat 
               
               
                 46441 
                 ccttgagtgt tctttaatct tttagcaatt ccttgttgca tgttaaaaga ttagttatat 
               
               
                 46501 
                 tttattcctc atttgtgttc gttttcacca ggaggctcaa ttcaggcttc tttgcttact 
               
               
                 46561 
                 tggtgtctct agttctggtg cctggtgctt tggtcaatga agtggggttg gtaggattct 
               
               
                 46621 
                 attacttacc tgttttttgg ttttattttt tgttttgcag  TTCTCCGGGA GATGTTGCAT   
               
               
                 
                   46681 
                 
                 
                   AACCACTCCT TCGTGGGCTG TGTGAATCCT CAGTGGGCCT TGGCACAGCA TCAAACCAAG 
                 
               
               
                 
                   46741 
                 
                   TTATACCTTC TCAACACCAC CAAGCTTAG g taaatcagct gagtgtgtga acaagcagag 
               
               
                 46801 
                 ctactacaac aatggtccag ggagcacagg cacaaaagct aaggagagca gcatgaggta 
               
               
                 46861 
                 gttgggaggg cacaggcttt ggagtcagac acatgtggtt tcaaatccaa gttcgaccat 
               
               
                 46921 
                 ttcccattta tttgactgta gacaagttac attcctaaac tatgtctcag atttctcatc 
               
               
                 46981 
                 tgtaagttgt ggtattacta gttaacatgc aggggttttg tttgtttgtt tgtttgtttg 
               
               
                 47041 
                 tttgtgaggg taagaaataa cccaagaagc ctagtccttg gtagttgctc agtgccctat 
               
               
                 47101 
                 aaatgttgtg aaccaggtgg tgagggtttg gtgctgctag agaattctgg tatctgctct 
               
               
                 47161 
                 gtgcaacaga gtactgtagg tgatgcaaga gaaagaagac ctgatgcctt ctttcctccc 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 116) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-14A-s: 
                 5′ (*)-GGTCAATGAAGTGGGG 3′ 
                 (SEQ ID NO.: 117) 
                   
               
               
                   
               
               
                 MLH1-14A-as: 
                 5′ CCACGAAGGAGTGGTTA 3′ 
                 (SEQ ID NO.: 118) 
               
               
                   
               
               
                 MLH1-14B-s: 
                 5′ AGTTCTCCGGGAGATG 3′ 
                 (SEQ ID NO.: 119) 
               
               
                   
               
               
                 MLH1-14B-as: 
                 5′ (*)-TACCTCATGCTGCTCTC 3′ 
                 (SEQ ID NO.: 120) 
               
               
                   
               
               
                 MLH1-14seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGTGTTCGTTTTCACCAGGAGG 
                 (SEQ ID NO.: 121) 
               
               
                   
               
               
                 MLH1-14seq-as: 
                 TCCCCAACCCCCTAAAGCGATCGAACTTGGATTTGAAACCAC 
                 (SEQ ID NO.: 122) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 15 
               
            
           
           
               
               
               
            
               
                 48301 
                 tttaggaaga ctccctgccc ttcctataca tttcacataa tttttaataa gttgtaaaaa 
                   
               
               
                 48361 
                 agtgatttat aggattcttt gtaagtgggg gaagttaagc agacaaaaag tttttaaatc 
               
               
                 48421 
                 ttactgcaga gtgtcaggaa ccttttatag caccagacag gtagggacag aacatgagtg 
               
               
                 48481 
                 gcagcaagcc agacttggtc ttagtgctct aacctgtctg ttagaggctg gccagtcaga 
               
               
                 48541 
                 cccctggttg aagacgttgg gaatcccagc tctttggagg ggtaagagat tttgttagac 
               
               
                 48601 
                 tgttaaccag attccacagc caggcagaac tatttctgtc tcatccatgt ttcagggatt 
               
               
                 48661 
                 acttctccca ttttgtccca actggttgta tctcaagcat gaattcagct tttccttaaa 
               
               
                 48721 
                 gtcacttcat ttttattttc ag TGAAGAAC TGTTCTACCA GATACTCATT TATGATTTTG   
               
               
                 
                   48781 
                 
                   CCAATTTTGG TGTTCTCAGG TTATCG gtaa gtttagatcc ttttcacttc tgaaatttca 
               
               
                 48841 
                 actgatcgtt tctgaaaata gtagctctcc actaatatct tatttgtagt atgttaaatt 
               
               
                 48901 
                 tttctaaaac ttctaaggat agttgctgta ttgtatgatt tgcatatgga ggtatctata 
               
               
                 48961 
                 agaagtttta tactttttag caaaatagtc atttggtagc caacttaaac aaatgtttat 
               
               
                 49021 
                 taatatagaa gttaataata tctactgata ctcggccggg tgcggtggct catgcctgta 
               
               
                 49081 
                 atcccaccac tttgggaggc tgaggcgggc agatcatttg aggtcaggag ttcaagacca 
               
               
                 49141 
                 gcctgaccaa tatgatgaaa ccctgtctct actaaattac aaatattagc agggtatggt 
               
               
                 49201 
                 ggtgggcgcc tgtaatccca gctactcagg aggctaaggc aggagaatca tttgaaccca 
               
               
                 49261 
                 ggaggcagag gttgcaatga gctgagatca cgccactgca ctccagcctg ggcaacagag 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 123) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-15-s: 
                 5′ TTCAGGGATTACTTCTC 3′ 
                 (SEQ ID NO.: 124) 
                   
               
               
                   
               
               
                 MLH1-15-as: 
                 5′ (*)-GAAAAATTTAACATACTACA 3′ 
                 (SEQ ID NO.: 125) 
               
               
                   
               
               
                 MLH1-15seq-s2: 
                 TCTGCCTTTTTCTTCCATCGGGAGATTCCACAGCCAGGCAG 
                 (SEQ ID NO.: 126) 
               
               
                   
               
               
                 MLH1-15seq-as2: 
                 TCCCCAACCCCCTAAAGCGATACCTCCATATGCAAATCATACAA 
                 (SEQ ID NO.: 127) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 16 
               
            
           
           
               
               
               
            
               
                 53581 
                 gcattagatg atttacctga aatgtcattc aatttaactt actctccatc ctcacccgcc 
                   
               
               
                 53641 
                 cagctttggt tatgaggcag tagaaagaaa tgatctgcct gtggttttct agaaatacga 
               
               
                 53701 
                 aagttgagtc cttaaggcta cacagaaaga aagtacctcc ccagggcttc acccttccca 
               
               
                 53761 
                 tcctttcagc aggctttttg tctgtcgtat cttctctgtt gaaatggcca ttgacaagag 
               
               
                 53821 
                 gaggaaaggg gttttgttgt ggattgttca ggcacttcct ttggggtata tgggggatga 
               
               
                 53881 
                 gtgttacatt tatggtttct cacctgccat tctgatagtg gattcttggg aattcaggct 
               
               
                 53941 
                 tcatttggat gctccgttaa agcttgctcc ttcatgttct tgcttcttcc tag GAGCCAG   
               
               
                 
                   54001 
                 
                 
                   CACCGCTCTT TGACCTTGCC ATGCTTGCCT TAGATAGTCC AGAGAGTGGC TGGACAGAGG 
                 
               
               
                 
                   54061 
                 
                 
                   AAGATGGTCC CAAAGAAGGA CTTGCTGAAT ACATTGTTGA GTTTCTGAAG AAGAAGGCTG 
                 
               
               
                 
                   54121 
                 
                   AGATGCTTGC AGACTATTTC TCTTTGGAAA TTGATGAG gt gtgacagcca ttcttatact 
               
               
                 54181 
                 tctgttgtat tcttcaaata aaatttccag ccgggtgcgg tggctcatgg ctgtaatccc 
               
               
                 54241 
                 agcactttgg gaggctgagg tgggcagata acttggggtc aggagttcaa aaccagctgg 
               
               
                 54301 
                 ccaacatgat gaaaccccgt ctctactaaa aaaatagaaa aattagccag gcgtggtggc 
               
               
                 54361 
                 gggtacctgt aatccaagct gctcaggagg ctgaggcaga agaatcactt aaacccaaga 
               
               
                 54421 
                 ggtagaagtt gcagtgagcc gagattgcac cactgcactc tagcctaggc gacagcgaga 
               
               
                 54481 
                 ctgcgtctca aaaaaaaaaa aaaagaacgt tccaaggtca ggactaggcc tcccctcaga 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 128) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-16A-s: 
                 5′ (*)-GCCATTCTGATAGTGGA 3′ 
                 (SEQ ID NO.: 129) 
                   
               
               
                   
               
               
                 MLH1-16A-as2: 
                 5′ TCTAAGGCAAGCATGGCAA 
                 (SEQ ID NO.: 130) 
               
               
                   
               
               
                 MLH1-16B-s: 
                 5′ GCACCGCTCTTTGA 3′ 
                 (SEQ ID NO.: 131) 
               
               
                   
               
               
                 MLH1-16B-as: 
                 5′ (*)-GTATAAGAATGGCTGTCA 3′ 
                 (SEQ ID NO.: 132) 
               
               
                   
               
               
                 MLH1-16C-s2: 
                 5′ GGCTGAGATGCTTGCAG 3′ 
                 (SEQ ID NO.: 133) 
               
               
                   
               
               
                 MLH1-16C-as2: 
                 5′ (*)-CATGAGCCACCGCAC 3′ 
                 (SEQ ID NO.: 134) 
               
               
                   
               
               
                 MLH1-16seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGGGTTTTGTTGTGGATTGTTCAGG 
                 (SEQ ID NO.: 135) 
               
               
                   
               
               
                 MLH1-16seq-as: 
                 TCCCCAACCCCCTAAAGCGATGGGATTACAGCCATGAGCC 
                 (SEQ ID NO.: 136) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 17 
               
            
           
           
               
               
               
            
               
                 54661 
                 gagccgaatc cctgcaggcc attataaatg agattatgcc atttgctccc atttcttctt 
                   
               
               
                 54721 
                 attctttcat ttttggggct ctccatcttg atgtgttctt tggatcgtga acagatccaa 
               
               
                 54781 
                 agaaaaggtt gttctgccgt gctgtttgtc aggatgaaaa actctttttt aagtgtttag 
               
               
                 54841 
                 gtctgccccc agtgcccagc ccaatcaagt aacgtggtca cccagagtgg cagataggag 
               
               
                 54901 
                 cacaaggcct gggaaagcac tggagaaatg ggatttgttt aaactatgac agcattattt 
               
               
                 54961 
                 cttgttccct tgtccttttt cctgcaagca g GAAGGGAAC CTGATTGGAT TACCCCTTCT   
               
               
                 
                   55021 
                 
                 
                   GATTGACAAC TATGTGCCCC CTTTGGAGGG ACTGCCTATC TTCATTCTTC GACTAGCCAC 
                 
               
               
                 
                   55081 
                 
                   TGAG gtcagt gatcaagcag atactaagca tttcggtaca tgcatgtgtg ctggagggaa 
               
               
                 55141 
                 agggcaaatg accacccttt gatctggaat gataaagatg ataagggtgg gatagctgaa 
               
               
                 55201 
                 ggcctgctct catccccact aatattcatt cccagcaata ttcagcagtc ccatttacag 
               
               
                 55261 
                 ttttaacgcc taaagtatca catttcgttt tttagcttta agtagtctgt gatctccgtt 
               
               
                 55321 
                 tagaatgaga atgtttaaat tcgtacctat tttgaggtat tgaatttctt tggaccaggt 
               
               
                 55381 
                 gaattgggac gaagaaaagg aatgttttga aagcctcagt aaagaatgcg ctatgttcta 
               
               
                 55441 
                 ttccatccgg aagcagtaca tatctgagga gtcgaccctc tcaggccagc aggtacagtg 
               
               
                 55501 
                 gtgatgcaca ctggcacccc aggactagga caggacctca tacaatcttt aggagatgaa 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 137) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-17-s: 
                 5′ (*)-TGTTTAAACTATGACAGCA 3′ 
                 (SEQ ID NO.: 138) 
                   
               
               
                   
               
               
                 MLH1-17-as: 
                 5′ TGGTCATTTGCCCTT 3′ 
                 (SEQ ID NO.: 139) 
               
               
                   
               
               
                 MLH1-17seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGTTTAAGTGTTTAGGTCTGCCCC 
                 (SEQ ID NO.: 140) 
               
               
                   
               
               
                 MLH1-17seq-as: 
                 TCCCCAACCCCCTAAAGCGAGCTATCCCACCCTTATCATCTTT 
                 (SEQ ID NO.: 141) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 18 
               
            
           
           
               
               
               
            
               
                 54661 
                 gagccgaatc cctgcaggcc attataaatg agattatgcc atttgctccc atttcttctt 
                   
               
               
                 54721 
                 attctttcat ttttggggct ctccatcttg atgtgttctt tggatcgtga acagatccaa 
               
               
                 54781 
                 agaaaaggtt gttctgccgt gctgtttgtc aggatgaaaa actctttttt aagtgtttag 
               
               
                 54841 
                 gtctgccccc agtgcccagc ccaatcaagt aacgtggtca cccagagtgg cagataggag 
               
               
                 54901 
                 cacaaggcct gggaaagcac tggagaaatg ggatttgttt aaactatgac agcattattt 
               
               
                 54961 
                 cttgttccct tgtccttttt cctgcaagca ggaagggaac ctgattggat taccccttct 
               
               
                 55021 
                 gattgacaac tatgtgcccc ctttggaggg actgcctatc ttcattcttc gactagccac 
               
               
                 55081 
                 tgaggtcagt gatcaagcag atactaagca tttcggtaca tgcatgtgtg ctggagggaa 
               
               
                 55141 
                 agggcaaatg accacccttt gatctggaat gataaagatg ataagggtgg gatagctgaa 
               
               
                 55201 
                 ggcctgctct catccccact aatattcatt cccagcaata ttcagcagtc ccatttacag 
               
               
                 55261 
                 ttttaacgcc taaagtatca catttcgttt tttagcttta agtagtctgt gatctccgtt 
               
               
                 55321 
                 tagaatgaga atgtttaaat tcgtacctat tttgaggtat tgaatttctt tggaccag GT   
               
               
                 
                   55381 
                 
                 
                   GAATTGGGAC GAAGAAAAGG AATGTTTTGA AAGCCTCAGT AAAGAATGCG CTATGTTCTA 
                 
               
               
                 
                   55441 
                 
                   TTCCATCCGG AAGCAGTACA TATCTGAGGA GTCGACCCTC TCAGGCCAGC AG gtacagtg 
               
               
                 55501 
                 gtgatgcaca ctggcacccc aggactagga caggacctca tacaatcttt aggagatgaa 
               
               
                 55561 
                 acttgcccat ctctaaaatt tcgggatttc tttgtaccca acaaggttca aacacaacag 
               
               
                 55621 
                 tcagctttta ttcatgattt ttacttccat ctgctgatgt agaacatacc tccagagtga 
               
               
                 55681 
                 cctcagaaat tgtcaaatgt gaaaacacaa gccatcacag tgagaaatgg gaggttgagt 
               
               
                 55741 
                 tagattgtct aaggctggag agtccatata ctcccactgt tagctctgaa gtgtgtagcc 
               
               
                 55801 
                 agtcttcaga ttctgggtca gttgcctcag tctctcttag cttttgcctt actctttatc 
               
               
                 55861 
                 cgaccactgc cctgccagga aaacaaggct ctataactcc tcttacaggt cagcttgaca 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 142) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-18A-s: 
                 5′ (*)-TGTGATCTCCGTTTAGAA 3′ 
                 (SEQ ID NO.: 143) 
                   
               
               
                   
               
               
                 MLH1-18A-as2: 
                 5′ CTGAGAGGGTCGACTCC 
                 (SEQ ID NO.: 144) 
               
               
                   
               
               
                 MLH1-18B-s3: 
                 (*)-TGCGCTATGTTCTATTCCA 3′ 
                 (SEQ ID NO.: 145) 
               
               
                   
               
               
                 MLH1-18B-as3: 
                 5′ GCCGCCCCCGCCCGCTAGTCCTGGGGTGCCA 3′ 
                 (SEQ ID NO.: 146) 
               
               
                   
               
               
                 MLH1-18seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGAAGATGATAAGGGTGGGATAGC 
                 (SEQ ID NO.: 147) 
               
               
                   
               
               
                 MLH1-18seq-as: 
                 TCCCCAACCCCCTAAAGCGACCGAAATTTTAGAGATGGGC 
                 (SEQ ID NO.: 148) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 19 
               
            
           
           
               
               
               
            
               
                 56461 
                 tacttcctac agttgccatc caaatatcag tcaggatcag acatgatgtt agctcctgct 
                   
               
               
                 56521 
                 acaataaaac cattttctcc ctgaatgaaa acaaaggttc cacaggagac agtcccacag 
               
               
                 56581 
                 agcagtggct tcttttcctc cctttaaaac ctcatgttgg ctggacacag tggctcacac 
               
               
                 56641 
                 ctgtaatccc agcattttag gaggctgagg tgggaagatg gcttaagccc aggagtttga 
               
               
                 56701 
                 ggctgtagag ctatgatcac accactgccc ttcagcctgg gtgacagagc aagaccttgt 
               
               
                 56761 
                 ctctaaataa acaaacaaac aaaaaatcct cttgtgttca ggcctgtggg atcccctgag 
               
               
                 56821 
                 aggctagccc acaagatcca cttcaaaagc cctagataac accaagtctt tccagaccca 
               
               
                 56881 
                 gtgcacatcc catcagccag gacaccagtg tatgttggga tgcaaacagg gaggcttatg 
               
               
                 56941 
                 acatctaatg tgttttccag  AGTGAAGTGC CTGGCTCCAT TCCAAACTCC TGGAAGTGGA   
               
               
                 
                   57001 
                 
                 
                   CTGTGGAACA CATTGTCTAT AAAGCCTTGC GCTCACACAT TCTGCCTCCT AAACATTTCA 
                 
               
               
                 
                   57061 
                 
                 
                   CAGAAGATGG AAATATCCTG CAGCTTGCTA ACCTGCCTGA TCTATACAAA GTCTTTGAGA 
                 
               
               
                 
                   57121 
                 
                   GGTGTTAA at atggttattt atgcactgtg ggatgtgttc ttctttctct gtattccgat 
               
               
                 57181 
                 acaaagtgtt gtatcaaagt gtgatataca aagtgtacca acataagtgt tggtagcact 
               
               
                 57241 
                 taagacttat acttgccttc tgatagtatt cctttataca cagtggattg attataaata 
               
               
                 57301 
                 aatagatgtg tcttaacata ATTTCTTATTTAATTTTATTATGTATATATTGTGTCAGTTCAG 
               
               
                   
                 ATGCCAAAAAGAGGTCTTGAACATGTCACAGGCTCTGATGGCACTGACCATGGAGAAAGCT 
               
            
           
           
               
               
            
               
                 (SEQ ID NO.: 149) 
                   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MLH1-19A-s: 
                 5′ CAAGTCTTTCCAGACCC 3′ 
                 (SEQ ID NO.: 150) 
                   
               
               
                   
               
               
                 MLH1-19A-as: 
                 5′ (*)-TGTATAGATCAGGCAGGT 3′ 
                 (SEQ ID NO.: 151) 
               
               
                   
               
               
                 MLH1-19C-s: 
                 5′ (*)-CAGAAGATGGAAATATCCTGC 3′ 
                 (SEQ ID NO.: 152) 
               
               
                   
               
               
                 MLH1-19C-as: 
                 5′ (need 8 GC&#39;s)-TGTATATCACACTTTGATACAACACT3′ 
                 (SEQ ID NO.: 153) 
               
               
                   
               
               
                 MLH1-19B-s4 
                 AAGCCTTGCGCTCACAC 
                 (SEQ ID NO.: 154) 
               
               
                   
               
               
                 MLH1-19B-as4 
                 (*)-AATAACCATATTTAACACCTCTCAA 
                 (SEQ ID NO.: 155) 
               
               
                   
               
               
                 MLH1-19seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGGCTATGATCACACCACTGCCC 
                 (SEQ ID NO.: 156) 
               
               
                   
               
               
                 MLH1-19seq-as: 
                 TCCCCAACCCCCTAAAGCGACCTCTTTTTGGCATCTGAACTG 
                 (SEQ ID NO.: 157) 
               
               
                   
               
            
           
           
               
               
            
               
                 hMSH2 
                   
               
               
                 genomic seq. and primers 
               
               
                   
               
            
           
           
               
               
            
               
                 5′upstream region 
                   
               
               
                 tgttttcgaatgagtgaatcatcaacgagtggatgaaacgataatgtggctaacaggcagcagtaaggagg 
               
               
                 ctgtgtagaataaacccgtaatcccgatgttggcagtttgcttagaaagaaaaagggaggcagtcggagag 
               
               
                 gggcacacgttttaacaaaatactgggaggaggaggaaggctagttttttttttgttttcaagtttccttc 
               
               
                 tgatgttactcccatgcttccgggcacattacgagctcagtgcctgccggaaatctcccacctggtggcaa 
               
               
                 cctacccttgcatacaccccacccaggggcttcaagccttgcagctgagtaaacacagaaaggagctctac 
               
               
                 taaggatgcgcgtctgcgggtttccgcgcgacctaggcgcaggcatgcgcagtagctaaagtcaccagcgt 
               
               
                 gcgcgggaagctgggccgcgtctgcttatgattggttgccgcggcagactcccacccaccgaaacgcagcc 
               
               
                 ctggaagctgattgggtgtggtcgccgtggccggacgccgctcgggggacgtgggaggggaggcgggaaac 
               
               
                 (SEQ ID NO.: 158) 
               
               
                   
               
               
                 Exon 1 
               
            
           
           
               
               
               
            
               
                 1 
                 ggcgggaaac agcttagtgg gtgtggggtc gcgcattttc ttcaaccagg aggtgaggag 
                   
               
               
                 61 
                 gtttcgac AT GGCGGTGCAG CCGAAGGAGA CGCTGCAGTT GGAGAGCGCG GCCGAGGTCG   
               
               
                 
                   121 
                 
                 
                   GCTTCGTGCG CTTCTTTCAG GGCATGCCGG AGAAGCCGAC CACCACAGTG CGCCTTTTCG 
                 
               
               
                 
                   181 
                 
                 
                   ACCGGGGCGA CTTCTATACG GCGCACGGCG AGGACGCGCT GCTGGCCGCC CGGGAGGTGT 
                 
               
               
                 
                   241 
                 
                   TCAAGACCCA GGGGGTGATC AAGTACATGG GGCCGGCAG g tgagggccgg gacggcgcgt 
               
               
                 301 
                 gctggggagg gacccggggc cttgtggcgc ggctcctttc ccgcctcaga gagtgggcgg 
               
               
                 361 
                 tgagcagcct ctccagtgcg gaggcacggg ggcggaacgt tggtgcttgt gcggattccg 
               
               
                 421 
                 ccgtccccag gttctgcttg gctccggagg gacgcccccc tcagccctga aacccgtgcc 
               
               
                 481 
                 tctccagccg ccccggatct gaacttgtga tcacggagtg tttacgtcgt gccaggcatt 
               
               
                 541 
                 ttaatgcatt gttctagttc attttccagc agtcgcattc ctcgccttgg ccctacatgt 
               
               
                 601 
                 agcgctcatt acaaacacgg ccagaatctc ttattaacaa acagcagcca ggagtgagat 
               
               
                 661 
                 ttaaaataga ctgggggttt aggagaccct tttatgacac gtaattctgc tcccacgacg 
               
               
                 721 
                 ctcccattta taccgccggt ccagctaagg gtctggtaat ggagcgccgt tgaagagcag 
               
               
                 781 
                 tatgatgaag tggtcaggac caacggactc tggagctggg ctgcttggga tcaagtcgct 
               
               
                 841 
                 gcccctctgc ttattaacgt gtgaccttgg gccagtcatg gacgctatct gcttcagctc 
               
               
                 901 
                 agcattcagt gctctccgtc acccgacccc atctatccag gattatctct ccctggaaag 
               
               
                 961 
                 ctacaaacgt ctcaccctat gtgggccaaa tgttctggat aggcctagtt aacctcttct 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 159) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-1seq-s 
                 TCTGCCTTTTTCTTCCATCGGGGGCGGGAAACAGCTTAGTGG 
                 (SEQ ID NO.: 160) 
               
               
                   
               
               
                 MSH2-1seq-as 
                 TCCCCAACCCCCTAAAGCGACGCACTGGAGAGGCTGCTCA 
                 (SEQ ID NO.: 161) 
               
               
                   
               
            
           
           
               
               
            
               
                 ALTERNATIVE FCTL SEQ PRIMER SET: 
                   
               
            
           
           
               
               
               
               
            
               
                 MSH2-1seq-s2 
                 TCTGCCTTTTTCTTCCATCGGGGCGCAGTAGCTAAAGTCACCAG 
                 (SEQ ID NO.: 162) 
                   
               
               
                   
               
               
                 MSH2-1seq-as2 
                 TCCCCAACCCCCTAAAGCGAGAATCCGCACAAGCACCAAC 
                 (SEQ ID NO.: 163) 
               
               
                   
               
               
                 Exon 2 
               
            
           
           
               
               
               
            
               
                 4921 
                 gaattcccat gtattgtggg agggacctgg tgggagatag ttgaatcatg gggatggatc 
                   
               
               
                 4981 
                 tttcccatgc tgttgtgata gtgaataagc ctcatgagat ctgatggttt taaaaacgga 
               
               
                 5041 
                 agtctacctg cacaagctct ttctttgcct gctgccatcc atgtaagaca tgacttgttc 
               
               
                 5101 
                 ctccttgcct tctgccatga ttgtgagacc tccccagcca tgtggaacta taagtccagt 
               
               
                 5161 
                 aagcctcttt ttcttcccag tctcgggtat gtctttatca gcagcatgaa gtccagctaa 
               
               
                 5221 
                 tacagtgctt gaacatgtaa tatctcaaat ctgtaatgta cttttttttt ttttaagGAG 
               
               
                 
                   5281 
                 
                 
                   CAAAGAATCT GCAGAGTGTT GTGCTTAGTA AAATGAATTT TGAATCTTTT GTAAAAGATC 
                 
               
               
                 
                   5341 
                 
                 
                   TTCTTCTGGT TCGTCAGTAT AGAGTTGAAG TTTATAAGAA TAGAGCTGGA AATAAGGCAT 
                 
               
               
                 
                   5401 
                 
                   CCAAGGAGAA TGATTGGTAT TTGGCATATA AG gtaattat cttccttttt aatttactta 
               
               
                 5461 
                 tttttttaag agtagaaaaa taaaaatgtg aagaatttaa ttgtgtttta gtattttaag 
               
               
                 5521 
                 tagattgtga tagtagaatg gtttgagaca ctttaatagc aattagcatg tggtttttaa 
               
               
                 5581 
                 aaagttgcag tttggctggt cgcagtggct catgcttgta atcccagtat tttgggaggc 
               
               
                 5641 
                 tgaggcaggt aggttgcctg agcccaggag ttcaagacca gcctgcccaa cgtggtaaag 
               
               
                 5701 
                 ccccatctct actgaagata aaaaaattta aaaaaattag ctggggctat tggcacacac 
               
               
                 5761 
                 ctgtggtccc agctaatcaa gaggatgagg ttagaggatc acttgagccc aggaggttga 
               
               
                 5821 
                 ggttacagtt taactttcag aggccaaggc aggaggattg cttgagtcca ggagtttgag 
               
               
                 5881 
                 accaccctgg ggaatgtagg gagatcccat ctctatagag ggatagatta gatagataat 
               
               
                 5941 
                 ttctgagggg aggggagggg gagggccagg gaaggggagg gaaaggggag gggagggcag 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 164) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-2C-s: 
                 5′ ATAAGGCATCCAAGGAGAA 3′ 
                 (SEQ ID NO.: 165) 
               
               
                   
               
               
                 MSH2-2C-as: 
                 5′ (*)-ATCTACTTAAAATACTAAAACACAAT 3′ 
                 (SEQ ID NO.: 166) 
               
               
                   
               
               
                 MSH2-2B-s3 
                 (*)-GGAGCAAAGAATCTGCAGAG 
                 (SEQ ID NO.: 167) 
               
               
                   
               
               
                 MSH2-2B-as3 
                 TAATTACCTTATATGCCAAATACCA 
                 (SEQ ID NO.: 168) 
               
               
                   
               
               
                 MSH2-2seq-s2 
                 TCTGCCTTTTTCTTCCATCGGGTGCTGCCATCCATGTAAGAC 
                 (SEQ ID NO.: 169) 
               
               
                   
               
               
                 MSH2-2seq-as2 
                 TCCCCAACCCCCTAAAGCGACCAGCCAAACTGCAACTTTT 
                 (SEQ ID NO.: 170) 
               
               
                   
               
            
           
           
               
               
            
               
                 ALTERNATIVE FCTL SEQ PRIMER SET: 
                   
               
            
           
           
               
               
               
               
            
               
                 MSH2-2seq-s3 
                 TCTGCCTTTTTCTTCCATCGGGTTCCTCCTTGCCTTCTGCCAT 
                 (SEQ ID NO.: 171) 
                   
               
               
                   
               
               
                 MSH2-2seq-as3 
                 TCCCCAACCCCCTAAAGCGAGGGATTACAAGCATGAGCCACTG 
                 (SEQ ID NO.: 172) 
               
               
                   
               
            
           
           
               
               
            
               
                 Exon 3 
                   
               
               
                 ccctggttcaagcttttctcccgcctcagcctcccgagtagctgggattacaggtgcatgctgcaacaccc 
               
               
                 ggctaatttttgtatttttagtagagatggggtttcaccatgttggccaggacggtctcgatctcctgacc 
               
               
                 tcgtgatccgcctgccttggcctcccaaagtgttgggattacaggcgtgagccacagcactcagccagtta 
               
               
                 tttttttataagaaaacattttactggccaggcctggtggctcacacctgtaatcccagcactttgggagg 
               
               
                 ccgaggcaggcggatcacgaggtcaggagttcgagaccagcctggccaacatggtgaaaccccatctctac 
               
               
                 taaaaatacaaaaattagccaggcgtggtggtgtgcgcctgtattcccagctactggggaggctgaagcag 
               
               
                 gagaatcgattgaacccttgaggcagaggttgcagtgagttgagatcgcaccattgcactctagcctgggt 
               
               
                 gacagagcaagacttcatctcaaaaaaaagagaaaacattttattaataaggttcatagagtttggatttt 
               
               
                 tcctttttgcttataaaattttaaagtatgttcaagagtttgttaaatttttaaaattttatttttactta 
               
               
                 g GCTTCTCCTGGCAATCTCTCTCAGTTTGAAGACATTCTCTTTGGTAACAATGATATGTCAGCTTCCATTG   
               
               
                 
                   GTGTTGTGGGTGTTAAAATGTCCGCAGTTGATGGCCAGAGACAGGTTGGAGTTGGGTATGTGGATTCCATA 
                 
               
               
                 
                   CAGAGGAAACTAGGACTGTGTGAATTCCCTGATAATGATCAGTTCTCCAATCTTGAGGCTCTCCTCATCCA 
                 
               
               
                   GATTGGACCAAAGGAATGTGTTTTACCCGGAGGAGAGACTGCTGGAGACATGGGGAAACTGAGACAG gtaa 
               
               
                 gcaaattgagtctagtgatagaggagattccaggcctaggaaaggctctttaattgacatgatactgtttc 
               
               
                 atttaaggaaaaataataaaaaaactcttttttttgtatctaattaaaataatgttctgatgtttacagaa 
               
               
                 actttgtatatttaattggacattagaacaagctgtttgttgtgtaagatttattttacctcagatctttt 
               
               
                 ctcccccctttcctttctgtcttgtgttccaaaagagtaattattacggtaaatattactgtaattatgga 
               
               
                 tttatcaaataagatgcagttctttagcattttttgataaatcgagtggaactttagcctgttattttact 
               
               
                 atttgttttattttaa   (SEQ ID NO.: 173) 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 MSH2-3A-s: 
                 5′ (*)-AACATTTTATTAATAAGGTTC 3′ 
                 (SEQ ID NO.: 174) 
                   
               
               
                   
               
               
                 MSH2-3A-as: 
                 5′ ATTGCCAGGAGAAGC 3′ 
                 (SEQ ID NO.: 175) 
               
               
                   
               
               
                 MSH2-3B-s2: 
                 5′ (*)-ATTTTTACTTAGGCTTCTCCTG 3′ 
                 (SEQ ID NO.: 176) 
               
               
                   
               
               
                 MSH2-3B-as2: 
                 5′ CAGTTTCCCCATGTCTCC 3′ 
                 (SEQ ID NO.: 177) 
               
               
                   
               
               
                 MSH2-3C-s: 
                 5′ AATGTGTTTTACCCGGAG 3′ 
                 (SEQ ID NO.: 178) 
               
               
                   
               
               
                 MSH2-3C-as: 
                 5′ (*)-CTTAAATGAAACAGTATCATGTC 3′ 
                 (SEQ ID NO.: 179) 
               
               
                   
               
               
                 MSH2-3seq-s4 
                 TCTGCCTTTTTCTTCCATCGGGGGTTCATAGAGTTTGGATTTTTCC 
                 (SEQ ID NO.: 180) 
               
               
                   
               
               
                 MSH2-3seq-as4 
                 TCCCCAACCCCCTAAAGCGACCTTAAATGAAACAGTATCATGTCAA 
                 (SEQ ID NO.: 181) 
               
               
                   
               
               
                 Exon 4 
               
            
           
           
               
               
               
            
               
                 7501 
                 gtggcttgct cctgtaatcc tagctacttg ggaggctgag gcaggagaat tgcttgaacc 
                   
               
               
                 7561 
                 tgggaggcag aggtagcagt gagccaagat cgtgtcaccg cattccatcc tgggcgacag 
               
               
                 7621 
                 tgagactctg tctcaaaaca aaaaaagagt tgttaccgtt gggactattt tttgaaagct 
               
               
                 7681 
                 ttatgtgaac gtaattttat attttgatga aaatttagtt tattgatgta aaaagtgtat 
               
               
                 7741 
                 cagtacatca tatcagtgtc ttgcacattg tataaacatt taatgtaggt gaatctgtta 
               
               
                 7801 
                 tcactatagt tatcaatgtt ataattttca tttttgcttt tcttattcct tttctcatag 
               
               
                 7861 
                 tagtttaaac tatttctttc aaaatag ATA ATTCAAAGAG GAGGAATTCT GATCACAGAA   
               
               
                 
                   7921 
                 
                 
                   AGAAAAAAAG CTGACTTTTC CACAAAAGAC ATTTATCAGG ACCTCAACCG GTTGTTGAAA 
                 
               
               
                 
                   7981 
                 
                   GGCAAAAAGG GAGAGCAGAT GAATAGTGCT GTATTGCCAG AAATGGAGAA TCAG gtacat 
               
               
                 8041 
                 ggattataaa tgtgaattac aatatatata atgtaaatat gtaatatata ataaataata 
               
               
                 8101 
                 tgtaaactat agtgactttt tagaaggata tttctgtcat atttatctca aaacctaaac 
               
               
                 8161 
                 tgtgtatcaa tgatattaag cttttttttt tttttgagac agagtttcac ttttgttgcc 
               
               
                 8221 
                 caggctggag tacaatggcg cgatcttggc tcaccacatc ctctgcctcc caggttcaag 
               
               
                 8281 
                 tgatcctcct gccttggcct cctgagtagc tgggattaca ggcatgtgcc accacgcctg 
               
               
                 8341 
                 gctcatcttt tttgtatttt tagtagagat ggggtttctc tatgttggtc aggctggtct 
               
               
                 8401 
                 caaactcctg aacctcaggt gatccgcccg cctcgggctt ccaaagcgct gagattgcag 
               
               
                 8461 
                 gcatgagcca ctgtgtctgg cctattttta tagtttatgt acttggaatt atataatata 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 182) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-4A-s: 
                 5′ (*)-TCCTTTTCTCATAGTAGTTTA 3′ 
                 (SEQ ID NO.: 183) 
               
               
                   
               
               
                 MSH2-4A-as: 
                 5′ TTGAGGTCCTGATAAATG 3′ 
                 (SEQ ID NO.: 184) 
               
               
                   
               
               
                 MSH2-4A-s2: 
                 5′ (*)-TTTCTTTCAAAATAGATAATTC 3′ 
                 (SEQ ID NO.: 185) 
               
               
                   
               
               
                 MSH2-4A-as2: 
                 5′ TTTTTGCCTTTCAACA 3′ 
                 (SEQ ID NO.: 186) 
               
               
                   
               
               
                 MSH2-4B-2s: 
                 5′ ATTTATCAGGACCTCAA 3′ 
                 (SEQ ID NO.: 187) 
               
               
                   
               
               
                 MSH2-4B-2as: 
                 5′ (*)-TGTAATTCACATTTATAATC 3′ 
                 (SEQ ID NO.: 188) 
               
               
                   
               
               
                 MSH2-4C-s: 
                 5′ ATTGCCAGAAATGGAG 3′ 
                 (SEQ ID NO.: 189) 
               
               
                   
               
               
                 MSH2-4C-as: 
                 5′ (*)-ACATATTTACATTATATATATTGT 3′ 
                 (SEQ ID NO.: 190) 
               
               
                   
               
               
                 MSH2-4seq-s2: 
                 TCTGCCTTTTTCTTCCATCGGGgcattccatcctgggcga 
                 (SEQ ID NO.: 191) 
               
               
                   
               
               
                 MSH2-4seq-as2: 
                 TCCCCAACCCCCTAAAGCGACAGCCTGGGCAACAAAAGTG 
                 (SEQ ID NO.: 192) 
               
               
                   
               
               
                 Exon 5 
               
            
           
           
               
               
               
            
               
                 9361 
                 agagacgggg tttcactatg ttggctaggc tggtctcaaa ctcctagcct cgagtcatcc 
                   
               
               
                 9421 
                 acccgcctcg tcctcccgga gtgcttggat tacagcatga gccactgcgc ccggccccca 
               
               
                 9481 
                 ttttagtttt gatggacatt tgggtaattt tcttttttgg ctattctaaa taatgctgca 
               
               
                 9541 
                 attactgtta attttcacct tgtaaaaacc attttcaaat ctcaagagat taacctttag 
               
               
                 9601 
                 ttttcttggt ttggattggg aaggaacacc aaggaaaatg agggacttca gaatttattt 
               
               
                 9661 
                 tcattttgca tttgtttttt aaaatcttta gaactggatc cagtggtata gaaatcttcg 
               
               
                 9721 
                 atttttaaat tcttaatttt ag GTTGCAGT TTCATCACTG TCTGCGGTAA TCAAGTTTTT   
               
               
                 
                   9781 
                 
                 
                   AGAACTCTTA TCAGATGATT CCAACTTTGG ACAGTTTGAA CTGACTACTT TTGACTTCAG 
                 
               
               
                 
                   9841 
                 
                   CCAGTATATG AAATTGGATA TTGCAGCAGT CAGAGCCCTT AACCTTTTTC AG gtaaaaaa 
               
               
                 9901 
                 aaaaaaaaaa aaaaaaaaaa agggttaaaa atgttgaatg gttaaaaaat gttttcattg 
               
               
                 9961 
                 acatatactg aagaagctta taaaggagct aaaatatttt gaaatattat tatacttgga 
               
               
                 10021 
                 ttagataact agctttaaat ggctgtattt ttctctcccc tcctccactc cactttttaa 
               
               
                 10081 
                 cttttttttt tttaagtcag agtctcactt gttccctagg ccagagtgca gtggcacaat 
               
               
                 10141 
                 ctcagcccac tctaacctcc acctcccaag tagttgggat tacagttgcc tgccaccatg 
               
               
                 10201 
                 cctggttaat ttttatattt ttagtagggt tgcggggaca gggtttcacc atgttggcca 
               
               
                 10261 
                 ggttggtctc aaacttctga ccttaggtga tcctcccacc tcggcttccc aaagtgctgg 
               
               
                 10321 
                 gattacaggc ttgagccatc gtgcccagcc tactttttac ttttttagag actgggcttg 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 193) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-5A-s: 
                 5′ (*)-TTCATTTTGCATTTGTT 3′ 
                 (SEQ ID NO.: 194) 
               
               
                   
               
               
                 MSH2-5A-as: 
                 5′ CTTGATTACCGCAGAC 3′ 
                 (SEQ ID NO.: 195) 
               
               
                   
               
               
                 MSH2-5B-s: 
                 5′ (*)-ATCTTCGATTTTTAAATTC 3′ 
                 (SEQ ID NO.: 196) 
               
               
                   
               
               
                 MSH2-5B-as: 
                 5′ AAAGGTTAAGGGCTCTG 3′ 
                 (SEQ ID NO.: 197) 
               
               
                   
               
               
                 MSH2-5seq-s2: 
                 TCTGCCTTTTTCTTCCATCGGGTTCTTGGTTTGGATTGGGAAGG 
                 (SEQ ID NO.: 198) 
               
               
                   
               
               
                 MSH2-5seq-as2: 
                 TCCCCAACCCCCTAAAGCGAGGGGAGAGAAAAATACAGCCAT 
                 (SEQ ID NO.: 199) 
               
               
                   
               
            
           
           
               
               
            
               
                 ALTERNATIVE FCTL SEQ PRIMER SET: 
                   
               
            
           
           
               
               
               
               
            
               
                 MSH2-5seq-s3: 
                 TCTGCCTTTTTCTTCCATCGGGAGTTTTGATGGACATTTGGGTAA 
                 (SEQ ID NO.: 200) 
                   
               
               
                   
               
               
                 MSH2-5seq-as3: 
                 TCCCCAACCCCCTAAAGCGAGTTAAAAAGTGGAGTGGAGGAGG 
                 (SEQ ID NO.: 201) 
               
               
                   
               
               
                 Exon 6 
               
            
           
           
               
               
               
            
               
                 11101 
                 atggggtttc atcttgttgg ctaggctgga ctctaactcc aggtgatctg cctgcctcgg 
                   
               
               
                 11161 
                 cctcccaaat tgatgggatt acaggtgtaa accactgggc ctggcctagc aatttaaaat 
               
               
                 11221 
                 gacattctaa gaagttttat gtctaaatct gcagtaagtg gctgggtgac gtggctcatg 
               
               
                 11281 
                 cctgtaatcc caacgctttg ggagtccagg gtgggaggat gacttgaggc caggagttga 
               
               
                 11341 
                 gaccagcctg ggcaacatag tgagactctg tctctacaaa agaaaaaatt agcggggctt 
               
               
                 11401 
                 agtggcgtgc gcctgtagtc tcagctactc gaaaggctga agtgggagga ttctttgagc 
               
               
                 11461 
                 cccaagggtt ctggcttgcc gtgagccagg atggcaccac tgcactccag tctgggcaat 
               
               
                 11521 
                 agagtcagac cctgtctcaa caaataaaat aaaactgtag taattataaa gtggttttgg 
               
               
                 11581 
                 ctgggggaga aatgtacagt tgaacatacg gattaagagg ttgaaagttg gtcttaggaa 
               
               
                 11641 
                 gaggaacttt ttgtggaaat ttcttaatat ttgaagaata ttatgttatt gttcctctgt 
               
               
                 11701 
                 ttttcatggc gtagtaaggt tttcactaat gagcttgcca ttctttctat tttatttttt 
               
               
                 11761 
                 gtttactag G GTTCTGTTGA AGATACCACT GGCTCTCAGT CTCTGGCTGC CTTGCTGAAT   
               
               
                 
                   11821 
                 
                 
                   AAGTGTAAAA CCCCTCAAGG ACAAAGACTT GTTAACCAGT GGATTAAGCA GCCTCTCATG 
                 
               
               
                 
                   11881 
                 
                   GATAAGAACA GAATAGAGGA GAG gtatgtt attagtttat actttcgtta gttttatgta 
               
               
                 11941 
                 acctgcagtt acccacatga ttataccact tattgtaata tgcagttttg gaagtatatg 
               
               
                 12001 
                 ttaccattta actgtacaga gtacatagta atagagtggt aattatttag attgattaaa 
               
               
                 12061 
                 gaactcattt ttttaaataa gttttttttt tttcactata aaagtttatt ttatttgaga 
               
               
                 12121 
                 tggtatggta tcgaacatgt tcatattgtg tgtaatcgtg ggtaaattac tcaaccttta 
               
               
                 12181 
                 tgtcatagtt tcttcacctt taaaatgaca ttaataaaag agctacttaa taggattata 
               
               
                 12241 
                 agcatgagat gatttaatat acataaaata cttacagtct gatatatagg aagcacttaa 
               
               
                 12301 
                 ctctttatcc tagaaaagat ttaaggtgac cttaacatat atgtcagaaa atctttaaaa 
               
               
                 12361 
                 ttgtggaaat aaaaggttgt ataattctgc tatcctaaaa ttactagtat ttcaatatat 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 202) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-6A-s: 
                 5′ (*)-GTTTTTCATGGCGTAG 3′ 
                 (SEQ ID NO.: 203) 
               
               
                   
               
               
                 MSH2-6A-as: 
                 5′ ACTGAGAGCCAGTGGTA 3′ 
                 (SEQ ID NO.: 204) 
               
               
                   
               
               
                 MSH2-6B-s2: 
                 5′ TTTACTAGGGTTCTGTTGAAGA 
                 (SEQ ID NO.: 205) 
               
               
                   
               
               
                 MSH2-6B-as: 
                 5′ (*)-ATACCTCTCCTCTATTCTG 3′ 
                 (SEQ ID NO.: 206) 
               
               
                   
               
               
                 MSH2-6C-s: 
                 5′ TCAAGGACAAAGACTTGT 3′ 
                 (SEQ ID NO.: 207) 
               
               
                   
               
               
                 MSH2-6C-as: 
                 5′ (*)-CATATTACAATAAGTGGTATAAT 3′ 
                 (SEQ ID NO.: 208) 
               
               
                   
               
               
                 MSH2-6seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGTGAACATACGGATTAAGAGG 
                 (SEQ ID NO.: 209) 
               
               
                   
               
               
                 MSH2-6seq-as: 
                 TCCCCAACCCCCTAAAGCGACATATACTTCCAAAACTGCA 
                 (SEQ ID NO.: 210) 
               
               
                   
               
               
                 Exon 7 
               
            
           
           
               
               
               
            
               
                 24181 
                 ttttttttga gacagagtct tgctcttgtt gcccaggctg gagtgccatg gcatgatctc 
                   
               
               
                 24241 
                 agtgcaccac aatctctgct tcccaggttt aagcgattct cctgcctcag cctcccaagt 
               
               
                 24301 
                 agatgggatc acaggcatga gccaccatgc ctggctaatt ttgtattttt tgtacagacg 
               
               
                 24361 
                 gggtttctcc atgttggtca ggccagtctc gaactcccta cctcaggtga tctgcctgcc 
               
               
                 24421 
                 tcggcctctc aaagtgctgg gattacaggt gtgagccact gcgcccagca gattcaagct 
               
               
                 24481 
                 ttttaaatgg aattttgagc tgatttagtt gagacttacg tgcttagttg ataaatttta 
               
               
                 24541 
                 attttatact aaaatatttt acattaattc aagttaattt atttcag ATT GAATTTAGTG   
               
               
                 
                   24601 
                 
                 
                   GAAGCTTTTG TAGAAGATGC AGAATTGAGG CAGACTTTAC AAGAAGATTT ACTTCGTCGA 
                 
               
               
                 
                   24661 
                 
                 
                   TTCCCAGATC TTAACCGACT TGCCAAGAAG TTTCAAAGAC AAGCAGCAAA CTTACAAGAT 
                 
               
               
                 
                   24721 
                 
                 
                   TGTTACCGAC TCTATCAGGG TATAAATCAA CTACCTAATG TTATACAGGC TCTGGAAAAA 
                 
               
               
                 
                   24781 
                 
                   CATGAAG gta acaagtgatt ttgttttttt gttttccttc aactcataca atatatactt 
               
               
                 24841 
                 ggcaatgtgc tgtcctcata aagttggtgg tggtgactca ctcttaggac acattcagat 
               
               
                 24901 
                 ttcttttttt tttttttttg agaaggagtc ttgctccgtt gccaaggcta gagtgcagtg 
               
               
                 24961 
                 gcacaatctc agctcactgc aacctctgcc tcctgggttc aagcgattct cctgcctcag 
               
               
                 25021 
                 cttcctgagt ggctgggatt acaggcatgt gccaccatgc ccggctaatt tttgtacttt 
               
               
                 25081 
                 tagttttacc atgttggcca ggttcgtctg gaactcccaa tctcaggtga cccacctgcc 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 211) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-7A-s: 
                 5′ (*)-GTTGAGACTTACGTGCTT 3′ 
                 (SEQ ID NO.: 212) 
               
               
                   
               
               
                 MSH2-7A-as2: 
                 5′ CAATTCTGCATCTTCTACAAA 
                 (SEQ ID NO.: 213) 
               
               
                   
               
               
                 MSH2-7B-s2: 
                 5′ (*)-ATTTCAGATTGAATTTAGTGG 3′ 
                 (SEQ ID NO.: 214) 
               
               
                   
               
               
                 MSH2-7B-as2: 
                 5′ AGTTTGCTGCTTGTCTTTG 3′ 
                 (SEQ ID NO.: 215) 
               
               
                   
               
               
                 MSH2-7C-s3: 
                 5′ GACTTGCCAAGAAGTTT 3′ 
                 (SEQ ID NO.: 216) 
               
               
                   
               
               
                 MSH2-7C-as2: 
                 5′ (*)-TGAGTCACCACCACCAAC 3′ 
                 (SEQ ID NO.: 217) 
               
               
                   
               
               
                 MSH2-7seq-s3: 
                 TCTGCCTTTTTCTTCCATCGGGGCTGATTTAGTTGAGACTTACGTGC 
                 (SEQ ID NO.: 218) 
               
               
                   
               
               
                 MSH2-7seq-as2: 
                 TCCCCAACCCCCTAAAGCGAGAGGACAGCACATTGCCAAG 
                 (SEQ ID NO.: 219) 
               
               
                   
               
            
           
           
               
            
               
                 Exon 8 
               
            
           
           
               
               
               
            
               
                 40081 
                 tataagaaat gaaattcatt tagtcataat taatgtcatg tttctgcatc tatattactt 
                   
               
               
                 40141 
                 gttgggttta cagacgaggt agtgtattat tagtgggaag ctttgagtgc tacatcatct 
               
               
                 40201 
                 ccctttctat aaaataaatt gagtacgaaa caatttgaat taaaacacct gagtaaatag 
               
               
                 40261 
                 taactttgga gacctgctgt actatttgta ccttttggat caaatgatgc ttgtttatct 
               
               
                 40321 
                 cagtcaaaat tttatgattt gtattctgta aaatgagatc tttttatttg tttgttttac 
               
               
                 40381 
                 tactttcttt tag GAAAACA CCAGAAATTA TTGTTGGCAG TTTTTGTGAC TCCTCTTACT   
               
               
                 
                   40441 
                 
                 
                   GATCTTCGTT CTGACTTCTC CAAGTTTCAG GAAATGATAG AAACAACTTT AGATATGGAT 
                 
               
               
                 
                   40501 
                 
                 CAGgtatgca atatactttt taatttaagc agtagttatt tttaaaaagc aaaggccact 
               
               
                 40561 
                 ttaagaaagt ttgtagattt ttctttttag tatctaattg tagcaccttt gtggacagtg 
               
               
                 40621 
                 gatgtaatat taagtgacag atgggaaaag gatttttaaa aaaatagcaa ctgtttcagt 
               
               
                 40681 
                 ggatgaaata aagattatta gcagagaaaa tgaatattgg gcataactgt cctggtgaaa 
               
               
                 40741 
                 gacaatctca taaatgaaca atttcataat ttcgtaaatg caactgcatt ttattttcaa 
               
               
                 40801 
                 agagaaggaa aattatagtc actggaaacg gaaagagaag ttagaggtaa acataggaca 
               
               
                 40861 
                 cacaagaaaa ctttcatttt gtttattttc ttgtttttct tttgagacag ggtttccctc 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 220) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-8A-s: 
                 5′ (*)-TTTGGATCAAATGATGC 3′ 
                 (SEQ ID NO.: 221) 
               
               
                   
               
               
                 MSH2-8A-as: 
                 5′ ATCAGTAAGAGGAGTCACA 3′ 
                 (SEQ ID NO.: 222) 
               
               
                   
               
               
                 MSH2-8B-s: 
                 5′ TTGTGACTCCTCTTACTG 3′ 
                 (SEQ ID NO.: 223) 
               
               
                   
               
               
                 MSH2-8B-as: 
                 5′ (*)-AATAACTACTGCTTAAATTAA 3′ 
                 (SEQ ID NO.: 224) 
               
               
                   
               
               
                 MSH2-8C-s: 
                 5′ CTGACTTCTCCAAGTTTC 3′ 
                 (SEQ ID NO.: 225) 
               
               
                   
               
               
                 MSH2-8C-as: 
                 5′ GTGCTACAATTAGATACTAAA 3′ 
                 (SEQ ID NO.: 226) 
               
               
                   
               
               
                 MSH2-8D-s: 
                 5′ AGAAATTATTGTTGGCAGTT 
                 (SEQ ID NO.: 227) 
               
               
                   
               
               
                 MSH2-8D-as: 
                 5′ (*)-ATTGCATACCTGATCCATATC 
                 (SEQ ID NO.: 228) 
               
               
                   
               
               
                 MSH2-8seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGAATAGTAACTTTGGAGACCTGC 
                 (SEQ ID NO.: 229) 
               
               
                   
               
               
                 MSH2-8seq-as: 
                 TCCCCAACCCCCTAAAGCGACAGGACAGTTATGCCCAATA 
                 (SEQ ID NO.: 230) 
               
               
                   
               
               
                 Exon 9 
               
            
           
           
               
               
               
            
               
                 57541 
                 cacattgaac gttatttggt aatttttaga gaggacattt taaatgttta ggaaaaatat 
                   
               
               
                 57601 
                 aaataaaatg tagaatacta ttgggggcat atacatcatc agcactgtaa ctgtttcata 
               
               
                 57661 
                 tgaatcattt ttgtacatat agaactctaa agtcctaatg aacagaattt tacatttcta 
               
               
                 57721 
                 taaatagaaa gtccttaata gttgtgactg aataacttat ggatagcaaa ttatttaact 
               
               
                 57781 
                 gaaaacagta aaatttaagt gggaggaaat atttgcttta taatttctgt ctttacccat 
               
               
                 57841 
                 tatttatagg attttgtcac tttgttctgt ttgcag GTGG AAAACCATGA ATTCCTTGTA   
               
               
                 
                   57901 
                 
                 
                   AAACCTTCAT TTGATCCTAA TCTCAGTGAA TTAAGAGAAA TAATGAATGA CTTGGAAAAG 
                 
               
               
                 
                   57961 
                 
                   AAGATGCAGT CAACATTAAT AAGTGCAGCC AGAGATCTTG  gtaagaatgg gtcattggag 
               
               
                 58021 
                 gttggaataa ttcttttgtc tatacactgt atagacaaaa tattgatgcc agaattattt 
               
               
                 58081 
                 tataagttcc ctgtccccaa gatgatgact tcacatctct gtcaaacaga aatcgcccaa 
               
               
                 58141 
                 caggcccttg tatgatgtca tttaaacaag ccctatttta aatgtcacct ccactggtaa 
               
               
                 58201 
                 caggatactc ctaggaggat caccaagccc aattcttcta ggagtagtgc attgattagg 
               
               
                 58261 
                 ctttggggtt tccaagcagt tcattaatgt cacttttgga aaaagtctgt ctttcatacc 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 231) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-9-s2: 
                 5′ (*)-AATATTTGCTTTATAATTTC 3′ 
                 (SEQ ID NO.: 232) 
               
               
                   
               
               
                 MSH2-9-as2: 
                 5′ AGAATTATTCCAACCTC 3′ 
                 (SEQ ID NO.: 233) 
               
               
                   
               
               
                 MSH2-9seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGGAAAGTCCTTAATAGTTGTGACTG 
                 (SEQ ID NO.: 234) 
               
               
                   
               
               
                 MSH2-9seq-as: 
                 TCCCCAACCCCCTAAAGCGAGGGAACTTATAAAATAATTCTGGC 
                 (SEQ ID NO.: 235) 
               
               
                   
               
               
                 Exon 10 
               
            
           
           
               
               
               
            
               
                 61141 
                 tcatgcataa ctcctcgagg gtggggttac accttaatcc atcctcaggt gctcatggta 
                   
               
               
                 61201 
                 attggggcaa atatgttgcc cagtgctggt gctctgcagc cttggatggg tttacccaga 
               
               
                 61261 
                 aagcagcttt caagtcagaa actaacattc ataagggagt taaggatttt ataaatagat 
               
               
                 61321 
                 atccataatt catgtagttt tcaagtaagt agtatttgaa tcttttctgg ttagataata 
               
               
                 61381 
                 attgtgagta tgttgtcata taataacagt atgtttttca ctatttaaat aattttagaa 
               
               
                 61441 
                 ttacattgaa aaatggtagt aggtatttat ggaatacttt ttcttttctt cttgattatc 
               
               
                 61501 
                 aag GCTTGGA CCCTGGCAAA CAGATTAAAC TGGATTCCAG TGCACAGTTT GGATATTACT   
               
               
                 
                   61561 
                 
                 
                   TTCGTGTAAC CTGTAAGGAA GAAAAAGTCC TTCGTAACAA TAAAAACTTT AGTACTGTAG 
                 
               
               
                 
                   61621 
                 
                   ATATCCAGAA GAATGGTGTT AAATTTACCA ACAG gtttgc aagtcgttat tatattttta 
               
               
                 61681 
                 accctttatt aattccctaa atgctctaac atgatgtgaa tgttctatga taagttttac 
               
               
                 61741 
                 taatgtagtc atcaggtaag agtcaagctt tcttccatag agcagtcagc tgtcgcaaca 
               
               
                 61801 
                 ccatttgtta aatagtccgt ctgttctcca ttgactgaag tggtactttg ggtctatttt 
               
               
                 61861 
                 aaagactcta cttttacctc gtctcaccat tcttttgtct acacaaaata tattttatcg 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 236) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-10A-s: 
                 5′ (*)-GAATTACATTGAAAAATGG 3′ 
                 (SEQ ID NO.: 237) 
               
               
                   
               
               
                 MSH2-10A-as: 
                 5′ TTAATCTGTTTGCCAGG 3′ 
                 (SEQ ID NO.: 238) 
               
               
                   
               
               
                 MSH2-10B-s2: 
                 5′ TCTTCTTGATTATCAAGGC 3′ 
                 (SEQ ID NO.: 239) 
               
               
                   
               
               
                 MSH2-10B-as2: 
                 5′ (*)-ACACCATTCTTCTGGATA 3′ 
                 (SEQ ID NO.: 240) 
               
               
                   
               
               
                 MSH2-10C-s3: 
                 5′ TGCACAGTTTGGATATTACTT 3′ 
                 (SEQ ID NO.: 241) 
               
               
                   
               
               
                 MSH2-10C-as3: 
                 5′ (*)-GTAAAACTTATCATAGAACATTCAC 3′ 
                 (SEQ ID NO.: 242) 
               
               
                   
               
               
                 MSH2-10seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGTCATAAGGGAGTTAAGGATTT 
                 (SEQ ID NO.: 243) 494/536 
               
               
                   
               
               
                 MSH2-10seq-as: 
                 TCCCCAACCCCCTAAAGCGACTGCTCTATGGAAGAAAGCT 
                 (SEQ ID NO.: 244) 
               
               
                   
               
               
                 Exon 11 
               
            
           
           
               
               
               
            
               
                 65461 
                 gttctggggt tacaggcgtg agccaccacg cccggctgtc ttcaatctta aataaggatt 
                   
               
               
                 65521 
                 ccatttaaat attttgtaaa aggacacaga tcacagtttt actcagggga atataattgt 
               
               
                 65581 
                 tatagcagga attgtgccat tgcgctattc caaacagtgt aaaagaacat taataaattg 
               
               
                 65641 
                 aattctaact acatttgtcc ctaaggagtt gttcgttttc cacttgtatt tccattttaa 
               
               
                 65701 
                 ttatcattat ttggatgttt cataggatac tttggatatg tttcacgtag tacacattgc 
               
               
                 65761 
                 ttctagtaca cattttaata tttttaataa aactgttatt tcgatttgca g CAAATTGAC   
               
               
                 
                   65821 
                 
                 
                   TTCTTTAAAT GAAGAGTATA CCAAAAATAA AACAGAATAT GAAGAAGCCC AGGATGCCAT 
                 
               
               
                 
                   65881 
                 
                   TGTTAAAGAA ATTGTCAATA TTTCTTCAG g taaacttaat agaactaata atgttctgaa 
               
               
                 65941 
                 tgtcacctgg cttttggtaa cagaagaaaa atcatgatat ttgaagtgtg ttttgttatt 
               
               
                 66001 
                 ttcgcaagcc attacattct gactatttaa tatgttaggt ttcctatata aaataaggca 
               
               
                 66061 
                 tggtatgtta cagtaggaca cataactgga agttactctt gcacatagaa acaaaaaatg 
               
               
                 66121 
                 gcagaaaagc acaaaactta ctatagttgt aacagggaaa ggaaacacta gggcctacaa 
               
               
                 66181 
                 cgtactaatg tcttgggtca tctatgggct catgaggctc taggttatgg aagtaaatac 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 245) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-11A-s2: 
                 5′ TTTGGATATGTTTCACGTA 3′ 
                 (SEQ ID NO.: 246) 
               
               
                   
               
               
                 MSH2-11A-as2: 
                 5′ CTTTAACAATGGCATCCT 3′ 
                 (SEQ ID NO.: 247) 
               
               
                   
               
               
                 MSH2-11B-s2: 
                 5′ GCAAATTGACTTCTTTAAATG 3′ 
                 (SEQ ID NO.: 248) 
               
               
                   
               
               
                 MSH2-11B-as2: 
                 5′ ATGGCTTGCGAAAATAAC 3′ 
                 (SEQ ID NO.: 249) 
               
               
                   
               
               
                 MSH2-11seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGCATTTGTCCCTAAGGAGTTGTTC 
                 (SEQ ID NO.: 250) 
               
               
                   
               
               
                 MSH2-11seq-as: 
                 TCCCCAACCCCCTAAAGCGACAGAATGTAATGGCTTGCGA 
                 (SEQ ID NO.: 251) 
               
               
                   
               
               
                 Exon 12 
               
            
           
           
               
               
               
            
               
                 69361 
                 tgtggcgcaa tctcagctta ctgcaacttc caccttctgg gttcatgcaa ttctggtgcc 
                   
               
               
                 69421 
                 tcagcctccc aagtatctgg gtttacagac atgcaccacc atacctggct aatttttgta 
               
               
                 69481 
                 tttttggtag agatggggtt tcgccgtgtt accaggctgg tcttgaattc ctggccccat 
               
               
                 69541 
                 gtgatccccc ggcctcatgc gatctgcccg cctcagcctc cctaagtgct gggattatag 
               
               
                 69601 
                 gcgtgagcca cccaacccag ccagtactct gtttttgata gctattcaca atgggaaagg 
               
               
                 69661 
                 atgtagcaac acattttaac cctatgttga gttttaggtg ggttcctttg aaattttgtt 
               
               
                 69721 
                 aaggctaact tttgttaatt tttttaaaaa agtgtaaatt aggaaatggg ttttgaattc 
               
               
                 69781 
                 ccaaatgggg ggattaaatg tatttttacg gcttatatct gtttattatt cagtattcct 
               
               
                 69841 
                 gtgtacattt tctgttttta tttttataca g GCTATGTAG AACCAATGCA GACACTCAAT   
               
               
                 
                   69901 
                 
                 
                   GATGTGTTAG CTCAGCTAGA TGCTGTTGTC AGCTTTGCTC ACGTGTCAAA TGGAGCACCT 
                 
               
               
                 
                   69961 
                 
                 
                   GTTCCATATG TACGACCAGC CATTTTGGAG AAAGGACAAG GAAGAATTAT ATTAAAAGCA 
                 
               
               
                 
                   70021 
                 
                 
                   TCCAGGCATG CTTGTGTTGA AGTTCAAGAT GAAATTGCAT TTATTCCTAA TGACGTATAC 
                 
               
               
                 
                   70081 
                 
                   TTTGAAAAAG ATAAACAGAT GTTCCACATC ATTACTG gta aaaaacctgg tttttgggct 
               
               
                 70141 
                 ttgtgggggt aacgttttgt tttttttttt ttttttttaa tcttggagta gaaatatatt 
               
               
                 70201 
                 taaaattgat ggagaaaatt cccagttctt aacattagaa agggaatata ttattcttac 
               
               
                 70261 
                 cagttagtaa tctattcaca tttggtttag agggaagatt tagaaggtga gataaaagct 
               
               
                 70321 
                 tgtgagagaa tagtgtattc atgtgaaact tcttccatgg gttcagagca tttagaaaca 
               
               
                 70381 
                 aacatccctt cacactcaaa gcttaccttt gagccagtcc tccaatagtg aggtctttga 
               
               
                 70441 
                 aggtcaggcc aaattggctg tgggaggacc tcaggttagg ataggaatta ttttaagaca 
               
               
                 70501 
                 tggcactata ttcatgtgaa actcgcaaaa actagccttg catataggct catgtatcat 
               
               
                 70561 
                 gtctcagctg agatgtttga gagatcttaa ctagattcta gaaaacaaaa aaggaagtag 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 252) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-12A-s: 
                 5′ (*)-AGGAAATGGGTTTTGAA 3′ 
                 (SEQ ID NO.: 253) 
               
               
                   
               
               
                 MSH2-12A-as: 
                 5′ GAGCTAACACATCATTGAGT 3′ 
                 (SEQ ID NO.: 254) 
               
               
                   
               
               
                 MSH2-12B-s: 
                 5′ (*)-ATTTTTATACAGGCTATGTAG 3′ 
                 (SEQ ID NO.: 255) 
               
               
                   
               
               
                 MSH2-12B-as: 
                 5′ ACATATGGAACAGGTGCT 3′ 
                 (SEQ ID NO.: 256) 
               
               
                   
               
               
                 MSH2-12C-s: 
                 5′ TGGAGCACCTGTTCCAT 3′ 
                 (SEQ ID NO.: 257) 
               
               
                   
               
               
                 MSH2-12C-as: 
                 5′ (*)-AACAAAACGTTACCCCC 3′ 
                 (SEQ ID NO.: 258) 
               
               
                   
               
               
                 MSH2-12E-s: 
                 5′ CAGCTTTGCTCACGTGTCA 
                 (SEQ ID NO.: 259) 
               
               
                   
               
               
                 MSH2-12E-as: 
                 5′ (*)-CATCTTGAACTTCAACACAAGC 
                 (SEQ ID NO.: 260) 
               
               
                   
               
               
                 MSH2-12seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGTGTTGAGTTTTAGGTGGGTTCC 
                 (SEQ ID NO.: 261) 
               
               
                   
               
               
                 MSH2-12seq-as: 
                 TCCCCAACCCCCTAAAGCGATACCCCCACAAAGCCCAAA 
                 (SEQ ID NO.: 262) 
               
               
                   
               
               
                 Exon 13 
               
            
           
           
               
               
               
            
               
                 71041 
                 atgggcagta actctgtcca catctttggg caggctgtgg ttctgccttt atatgctatg 
                   
               
               
                 71101 
                 tcagtgtaaa cctacgcgat taatcatcag tgtacagttt aggactaaca atccatttat 
               
               
                 71161 
                 tagtagcaga aagaagttta aaatcttgct ttctgatata atttgttttg tag GCCCCAA   
               
               
                 
                   71221 
                 
                 
                   TATGGGAGGT AAATCAACAT ATATTCGACA AACTGGGGTG ATAGTACTCA TGGCCCAAAT 
                 
               
               
                 
                   71281 
                 
                 
                   TGGGTGTTTT GTGCCATGTG AGTCAGCAGA AGTGTCCATT GTGGACTGCA TCTTAGCCCG 
                 
               
               
                 
                   71341 
                 
                 
                   AGTAGGGGCT GGTGACAGTC AATTGAAAGG AGTCTCCACG TTCATGGCTG AAATGTTGGA 
                 
               
               
                 
                   71401 
                 
                   AACTGCTTCT ATCCTCAG gt aagtgcatct cctagtccct tgaagataga aatgtatgtc 
               
               
                 71461 
                 tctgtcctgt gagaaggaaa agtatatttg cagattctca tgtaaaaaca tctgagaatg 
               
               
                 71521 
                 tttgtcttag tttaatagtt gttttcctgt ggactttata tactttgtat tgtcttaaaa 
               
               
                 71581 
                 gagtgattga tggtagctac ggaaaacttt gatttttaaa attgtctctt taagtagaca 
               
               
                 71641 
                 atttataagc tactggtacg agttcacctt ataaatctcc actaccatgt ttttgcttgg 
               
               
                 71701 
                 actgttcaca cttcctggaa tggtccttct tgccgtttat ccaacttctt tctaattttt 
               
               
                 71761 
                 aagtccctaa tgatgggaat tctatttctg tagtgatttt tctggtcata cgaccgtaag 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 263) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-13A-s: 
                 5′ (*)-TAGGACTAACAATCCATT 3′ 
                 (SEQ ID NO.: 264) 
               
               
                   
               
               
                 MSH2-13A-as: 
                 5′ TGGGCCATGAGTACTA 3′ 
                 (SEQ ID NO.: 265) 
               
               
                   
               
               
                 MSH2-13B-s: 
                 5′ (*)-ATGGGAGGTAAATCAAC 3′ 
                 (SEQ ID NO.: 266) 
               
               
                   
               
               
                 MSH2-13B-as: 
                 5′ GACTCCTTTCAATTGACT 3′ 
                 (SEQ ID NO.: 267) 
               
               
                   
               
               
                 MSH2-13C-s4: 
                 5′ TTGTGGACTGCATCTTAGCC 
                 (SEQ ID NO.: 268) 
               
               
                   
               
               
                 MSH2-13C-5as: 
                 TCACAGGACAGAGACATACATTTC 
                 (SEQ ID NO.: 269) 
               
               
                   
               
               
                 MSH2-13seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGGCTATGTCAGTGTAAACCTACGC 
                 (SEQ ID NO.: 270) 
               
               
                   
               
               
                 MSH2-13seq-as: 
                 TCCCCAACCCCCTAAAGCGACTTCTCACAGGACAGAGACATACA 
                 (SEQ ID NO.: 271) 
               
               
                   
               
               
                 Exon 14 
               
            
           
           
               
               
               
            
               
                 72661 
                 ccgttgtttg ttcatgttca tgaccttttt ttttttttcc tattctcctc ccttcctccc 
                   
               
               
                 72721 
                 tccctccctc ccttccttcc ttccctcctt ccctccttcc ctccctccct cccacacaaa 
               
               
                 72781 
                 ggtgtgtgct accatacctg gctagttttt aatttttttt tttttttttt tttttagagg 
               
               
                 72841 
                 caaggtctca ctatgttgct caggctggtc tgggctcaag tgatcctccc acctccgcct 
               
               
                 72901 
                 tccaaagtgc tgggattaca gacgtgagcc atcatgcctg gcccttgccc atttttctat 
               
               
                 72961 
                 tgaagtttta gtgcttttta ttgactttgt ttatatatta agataatcca ttatgtttgt 
               
               
                 73021 
                 ggcatatcct tcccaatgta ttgtcttaat tttgtttttg tatgtgtatg ttaccacatt 
               
               
                 73081 
                 ttatgtgatg ggaaatttca tgtaattatg tgcttcag GT CTGCAACCAA AGATTCATTA   
               
               
                 
                   73141 
                 
                 
                   ATAATCATAG ATGAATTGGG AAGAGGAACT TCTACCTACG ATGGATTTGG GTTAGCATGG 
                 
               
               
                 
                   73201 
                 
                 
                   GCTATATCAG AATACATTGC AACAAAGATT GGTGCTTTTT GCATGTTTGC AACCCATTTT 
                 
               
               
                 
                   73261 
                 
                 
                   CATGAACTTA CTGCCTTGGC CAATCAGATA CCAACTGTTA ATAATCTACA TGTCACAGCA 
                 
               
               
                 
                   73321 
                 
                   CTCACCACTG AAGAGACCTT AACTATGCTT TATCAGGTGA AGAAAG gtat gtactattgg 
               
               
                 73381 
                 agtactctaa attcagaact tggtaatggg aaacttacta cccttgaaat catcagtaat 
               
               
                 73441 
                 tgccttattc taagttagta taaattattg atgttgttat agaacccatt taccccttaa 
               
               
                 73501 
                 ttcacagtct gggggtagga acatgtacat catatttctg tatctcatag taggaccact 
               
               
                 73561 
                 cattctaaag cattcacaga aagaattatc tgtactcttt ttgggacaga atctcgttct 
               
               
                 73621 
                 gttgcccagg ctggagtgcg atctcggctc actgcaacct ccgcctcccg ggttcaagcg 
               
               
                 73681 
                 attctcctgc ctcagcttcc cgagtagctg ggattacagg cgcctgccac cacacctggc 
               
               
                 73741 
                 taatttttat atttttagta gagacggggt ttcaccatgc tggccaggct ggtctcgaat 
               
               
                 73801 
                 tcctgacctc aggcaatcca cccgtctcgg cctcccaaag tgctgggatt acaggtgtga 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 272) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-14A-s3 
                 5′ (*)-GTATGTGTATGTTACCACATT 3′ 
                 (SEQ ID NO.: 273) 
               
               
                   
               
               
                 MSH2-14A-as3: 
                 5′ TAGTTAAGGTCTCTTCAGTG 3′ 
                 (SEQ ID NO.: 274) 
               
               
                   
               
               
                 MSH2-14B-s: 
                 5′ ATAATCTACATGTCACAGCA 3′ 
                 (SEQ ID NO.: 275) 
               
               
                   
               
               
                 MSH2-14B-as: 
                 5′ (*)-GAATAAGGCAATTACTGAT 3′ 
                 (SEQ ID NO.: 276) 
               
               
                   
               
               
                 MSH2-14seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGATGTTTGTGGCATATCCTTCC 
                 (SEQ ID NO.: 277) 
               
               
                   
               
               
                 MSH2-14seq-as: 
                 TCCCCAACCCCCTAAAGCGATAGTAAGTTTCCCATTACCAAGTTC 
                 (SEQ ID NO.: 278) 
               
               
                   
               
               
                 Exon 15 
               
            
           
           
               
               
               
            
               
                 75181 
                 ccctccctta ccttcccatg aaatgagaaa gcctcagaga tagtggcttg attaattttt 
                   
               
               
                 75241 
                 ctttagatta agatatttgt ctaagccttt aaggtttatc tattgagctt ttttgtctcc 
               
               
                 75301 
                 tatttttatt tttcctacta tgtttgtcga ggataaaata cagcactgtg tgccaagtca 
               
               
                 75361 
                 taatcacttt tcatttgaga cttaattaaa atgcctttat tttaatgata tatttggcta 
               
               
                 75421 
                 atgtatttga agtaatccga aattaagttt tctaatgaca aggtgagaag gataaattcc 
               
               
                 75481 
                 atttacataa attgctgtct cttctcatgc tgtcccctca cgcttcccca aatttcttat 
               
               
                 75541 
                 ag GTGTCTGT GATCAAAGTT TTGGGATTCA TGTTGCAGAG CTTGCTAATT TCCCTAAGCA   
               
               
                 
                   75601 
                 
                 
                   TGTAATAGAG TGTGCTAAAC AGAAAGCCCT GGAACTTGAG GAGTTTCAGT ATATTGGAGA 
                 
               
               
                 
                   75661 
                 
                   ATCGCAAGGA TATGATATCA TGGAACCAGC AGCAAAGAAG TGCTATCTGG AAAGAGAG gt 
               
               
                 75721 
                 ttgtcagttt gttttcatag tttaacttag cttctctatt attacataaa caggacacta 
               
               
                 75781 
                 agatgaaggt tttttgttgt tgtttgtttt cctctgtgtt tctagtgctt attttttaat 
               
               
                 75841 
                 cagttttttt gatggcaaag aatctatctc tgtgttattt tgatttctgc agtatataca 
               
               
                 75901 
                 tctgcatgat caatattcga tttcaagtac caaagtagga gtaaaggaat attaacctag 
               
               
                 75961 
                 gtttaaaatt agtcatttca ctaaaattag ttattatgga cgatagatgt ctaggtatat 
               
               
                 76021 
                 ctttgttcat aaacgaatat atcaagttca gttattaaat tacacattag gtaagaaaag 
               
               
                 76081 
                 gacaaagaaa taaaaaagca tgattcataa ttcctgccct ctatttgtct agaatttagt 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 279) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-15A-s 
                 5′ GTCTCTTCTCATGCTGTC 3′ 
                 (SEQ ID NO.: 280) 
               
               
                   
               
               
                 MSH2-15A-as 
                 5′ (*)-AATAGAGAAGCTAAGTTAAAC 3′ 
                 (SEQ ID NO.: 281) 
               
               
                   
               
               
                 MSH2-15seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGTTGGCTAATGTATTTGAAGTAATCC 
                 (SEQ ID NO.: 282) 
               
               
                   
               
               
                 MSH2-15seq-as: 
                 TCCCCAACCCCCTAAAGCGAACACAGAGGAAAACAAACAACAA 
                 (SEQ ID NO.: 283) 
               
               
                   
               
               
                 Exon 16 
               
            
           
           
               
               
               
            
               
                 77041 
                 gactctttta tgcaatctct tgtttccagt tagaatagaa gtcgtgtact tttgataaca 
                   
               
               
                 77101 
                 ttaattataa tatattttga gccctgtgag gttggtaaca ttattcccat tttatgaatg 
               
               
                 77161 
                 aggaatgtgt gttaaggagt ttgcccaaga gtcacatagc aagtcatagt catgctctct 
               
               
                 77221 
                 gaagcagcaa taacttggca ataaaataaa aatgaagcat cttctgtatg tgttaacttt 
               
               
                 77281 
                 tcagtgactg tttatgcctt ccagtattct ttgtaaacct tgaattcttt ttttcacaga 
               
               
                 77341 
                 tgattaaagt ttatcaattg taaaggtgga ggaatttggg aactagacag tgcacacata 
               
               
                 77401 
                 aataataaat atgttcttca aatattgggt gggctaatgt gggaggagtt tgagaccagc 
               
               
                 77461 
                 ctgggcaaca tagtgagacc ctcgtctcta aaaatatgaa aaataaaaaa aaaatttttt 
               
               
                 77521 
                 aaatgtgtga tatgtttaga tggaaatgaa acaatttgtc actgtctaac atgactttta 
               
               
                 77581 
                 gaaaagatat tttaattact aatgggacat tcacatgtgt ttcagCAAGG TGAAAAAATT 
               
               
                 77641 
                 ATTCAGGAGT TCCTGTCCAA GGTGAAACAA ATGCCCTTTA CTGAAATGTC AGAAGAAAAC 
               
               
                 77701 
                 ATCACAATAA AGTTAAAACA GCTAAAAGCT GAAGTAATAG CAAAGAATAA TAGCTTTGTA 
               
               
                 77761 
                 AATGAAATCA TTTCACGAAT AAAAGTTACT ACGTGAaaaa tcccagtaat ggaatgaagg 
               
               
                 77821 
                 taatattgat aagctattgt ctgtaatagt tttatattgt tttatattaa ccctttttcc 
               
               
                 77881 
                 atagtgttaa ctgtcagtgc ccatgggcta tcaacttaat aagatattta gtaatatttt 
               
               
                 77941 
                 actttgagga cattttcaaa gatttttatt ttgaaaaatg agagctgtaa ctgaggactg 
               
               
                 78001 
                 tttgcaattg acataggcaa taataagtga tgtgctgaat tttataaata aaatcatgta 
               
               
                 78061 
                 gtttgtgg 
               
            
           
           
               
               
               
               
            
               
                 (SEQ ID NO.: 284) 
                   
                   
                   
               
               
                   
               
               
                 MSH2-16A-s: 
                 5′ TTACTAATGGGACATTCACATG 3′ 
                 (SEQ ID NO.: 285) 
               
               
                   
               
               
                 MSH2-16A-as: 
                 5′ (*)-ACAATAGCTTATCAATATTACCTTC 3′ 
                 (SEQ ID NO.: 286) 
               
               
                   
               
               
                 MSH2-16seq-s: 
                 TCTGCCTTTTTCTTCCATCGGGGTAAAGGTGGAGGAATTTGGG 
                 (SEQ ID NO.: 287) 
               
               
                   
               
               
                 MSH2-16seq-as: 
                 TCCCCAACCCCCTAAAGCGAGGCACTGACAGTTAACACTATGGA 
                 (SEQ ID NO.: 288) 
               
               
                   
               
               
                 (*) = CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG (SEQ ID NO.: 344) 
               
            
           
         
       
     
     
       
         
           
               
               
             
               
                 TABLE B 
               
             
            
               
                   
               
               
                 MLH1 AND MSH2 TTGE PRIMERS 
                   
               
               
                 MLH1 TTGE Primers 3/3/04 AK 
               
               
                 OMIM 120436 
               
               
                 gene map 3p21.3, Locus ID 4292 
               
               
                 mRNA NM_000249 
               
               
                 earlier gonomic contig used AY_217549 
               
               
                 Reference numbering below refers to NC-000003, human chromosome 3. 
               
               
                 NC-000003 human chromosome 3. Region encompassing MLH1 gene from 36992890 . . . 37053065. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 start 
                   
                   
                   
                   
               
               
                 primer name 
                 SEQ ID 
                 start 
                 acc. 
                 end 
                 end acc. 
                 primer sequence 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 this is 5′-3′ sequence for each sense and 
                   
               
               
                 exon 1 
                   
                   
                   
                   
                   
                 antisense primer. (*) = GC clamp 
               
               
                 MLH1-1A-s: 
                 3 
                 2635 
                 36995524 
                 2650 
                 36995539 
                 5′ (*)-CAATAGCTGCCGCTGA 3′ 
               
               
                   
               
               
                 MLH1-1A-as: 
                 4 
                 2839 
                 36995728 
                 2854 
                 36995743 
                 5′ CGCTGGATAACTTCCC 3′ 
               
               
                   
               
               
                 MLH1-1B-s: 
                 5 
                 2834 
                 36995723 
                 2848 
                 36995737 
                 5′ GGCGGGGGAAGTTAT 3′ 
               
               
                   
               
               
                 MLH1-1B-as: 
                 6 
                 2944 
                 36995833 
                 2959 
                 36995848 
                 5′ (*)-CGCGCCATTGAGTGAC 3′ 
               
               
                   
               
               
                 MLH1-1C-s: 
                 7 
                 2870 
                 36995759 
                 2888 
                 36995777 
                 5′ (*)-CAAAGAGATGATTGAGAAC 3′ 
               
               
                   
               
               
                 MLH1-1C-as: 
                 8 
                 2975 
                 36995864 
                 2989 
                 36995878 
                 5′ CATGCCTCTGCCCGG 3′ 
               
               
                   
               
               
                 MLH1-1D-s: 
                 9 
                 2685 
                 36995574 
                 2702 
                 36995591 
                 5′ (*)-GGAAGAACGTGAGCACGA 3′ 
               
               
                   
               
               
                 MLH1-1D-as: 
                 10 
                 2850 
                 36995739 
                 2865 
                 36995754 
                 5′ CATTAGCTGGCCGCTG 3′ 
               
               
                   
               
               
                 exon 2 
               
               
                 MLH1-2A-s: 
                 16 
                 5765 
                 36998654 
                 5780 
                 36998669 
                 5′ (*)-TTATCATTGCTTGGCT 3′ 
               
               
                   
               
               
                 MLH1-2A-as: 
                 17 
                 5903 
                 36998792 
                 5920 
                 36998809 
                 5′ TTGTCTTGGATCTGAATC 3′ 
               
               
                   
               
               
                 MLH1-2B-s: 
                 18 
                 5853 
                 36998742 
                 5870 
                 36998759 
                 5′ (*)-GCAAAATCCACAAGTATT 3′ 
               
               
                   
               
               
                 MLH1-2B-as: 
                 19 
                 5974 
                 36998863 
                 5990 
                 36998879 
                 5′ CCTGACTCTTCCATGAA 3′ 
               
               
                   
               
               
                 exon 3 
               
               
                 MLH1-3A-s: 
                 23 
                 10115 
                 37003004 
                 10130 
                 37003019 
                 5′ (*)-GGGAATTCAAAGAGAT 3′ 
               
               
                   
               
               
                 MLH1-3A-as: 
                 24 
                 10283 
                 37003172 
                 10300 
                 37003189 
                 5′ TTCTTGAATCTTTAGCTT 3′ 
               
               
                   
               
               
                 MLH1-3B-s: 
                 25 
                 10195 
                 37003084 
                 10216 
                 37003105 
                 5′ ATATTGTATGTGAAAGGTTCAC 3′ 
               
               
                   
               
               
                 MLH1-3B-as: 
                 26 
                 10360 
                 37003249 
                 10381 
                 37003270 
                 5′ (*)-ACCAAACCTTATTTATCTATGT 3′ 
               
               
                   
               
               
                 exon 4 
               
               
                 MLH1-4A-s4 
                 32 
                 13562 
                 37006451 
                 13579 
                 37006468 
                 5′ GGTGAGGTGACAGTGGGT 3′ 
               
               
                   
               
               
                 MLH1-4A-as4 
                 33 
                 13710 
                 37006599 
                 13736 
                 37006625 
                 5′ (*)-TGAATATATATGAGTAAAAGAAGTCAG 3′ 
               
               
                   
               
               
                 MLH1-4B-s2 
                 34 
                 13654 
                 37006543 
                 13676 
                 37006565 
                 5′ TCATGTTACTATTACAACGAAAA 3′ 
               
               
                   
               
               
                 MLH1-4B-as2 
                 35 
                 13776 
                 37006665 
                 13796 
                 37006685 
                 5′ (*)-GATAACACTGGTGTTGAGACA 3′ 
               
               
                   
               
               
                 exon 5 
               
               
                 MLH1-5a-s: 
                 39 
                 16164 
                 37009053 
                 16182 
                 37009071 
                 5′ (*)-GGGATTAGTATCTATCTCT 3′ 
               
               
                   
               
               
                 MLH1-5A-as: 
                 40 
                 16234 
                 37009123 
                 16248 
                 37009137 
                 5′ GGCTTTCAGTTTTCC 3′ 
               
               
                   
               
               
                 MLH1-5B-s2: 
                 41 
                 16240 
                 37009129 
                 16255 
                 37009144 
                 5′ CTGAAAGCCCCTCCTA 3′ 
               
               
                   
               
               
                 MLH1-5B-as2: 
                 42 
                 16308 
                 37009197 
                 16327 
                 37009216 
                 5′ (*)-AGCTTCAACAATTTACTCTC 3′ 
               
               
                   
               
               
                 MLH1-5C-s2: 
                 43 
                 16273 
                 37009162 
                 16289 
                 37009178 
                 5′ CAAGGGACCCAGATCAC 3′ 
               
               
                   
               
               
                 MLH1-5C-as2: 
                 44 
                 16325 
                 37009214 
                 16346 
                 37009235 
                 5′ (*)-CCAATATTTATACAAACAAAGC 3′ 
               
               
                   
               
               
                 MLH1-5D-s 
                 45 
                 16197 
                 37009086 
                 16219 
                 37009108 
                 5′ (*)-TTTGTTATATTTTCTCATTAGAG 3′ 
               
               
                   
               
               
                 MLH1-5D-s 
                 46 
                 16281 
                 37009170 
                 16298 
                 37009187 
                 5′ ATTCTTACCGTGATCTGG 3′ 
               
               
                   
               
               
                 exon 6 
               
               
                 MLH1-6-5-s 
                 50 
                 17945 
                 37010834 
                 17967 
                 37010856 
                 5′ (*)-ATTCACTATCTTAAGACCTCGCT 3′ 
               
               
                   
               
               
                 MLH1-6-5-as 
                 51 
                 18168 
                 37011057 
                 18192 
                 37011081 
                 5′ CTAGAACACATTACTTTGATGACAA 3′ 
               
               
                   
               
               
                 exon 7 
               
               
                 MLH1-7-s: 
                 55 
                 20971 
                 37013860 
                 20986 
                 37013875 
                 5′ TAACTAAAAGGGGGCT 3′ 
               
               
                   
               
               
                 MLH1-7-as: 
                 56 
                 21191 
                 37014080 
                 21207 
                 37014096 
                 5′ (*)-TTTATTGTCTCATGGCT 3′ 
               
               
                   
               
               
                 exon 8 
               
               
                 MLH1-8A-s: 
                 60 
                 21157 
                 37014046 
                 21172 
                 37014061 
                 5′ (*)-GCTGGTGGAGATAAGG 3′ 
               
               
                   
               
               
                 MLH1-8A-as: 
                 61 
                 21278 
                 37014167 
                 21292 
                 37014181 
                 5′ TGTCCACGGTTGAGG 3′ 
               
               
                   
               
               
                 MLH1-8B-s: 
                 62 
                 21238 
                 37014127 
                 21258 
                 37014147 
                 5′ GGGGGCAAGGAGAGACAGTAG 3′ 
               
               
                   
               
               
                 MLH1-8B-as2: 
                 63 
                 21326 
                 37014215 
                 21345 
                 37014234 
                 5′ (*)-ATATAGGTTATCGACATACC 3′ 
               
               
                   
               
               
                 MLH1-8C-s2: 
                 64 
                 21312 
                 37014201 
                 21325 
                 37014214 
                 5′ AAATGCTGTTAGTC 3′ 
               
               
                   
               
               
                 MLH1-8C-as: 
                 65 
                 21397 
                 37014286 
                 21412 
                 37014301 
                 5′ (*)-TCTTGAAAGGTTCCAA 3′ 
               
               
                   
               
               
                 exon 9 
               
               
                 MLH1-9A-3-s 
                 69 
                 23605 
                 37016494 
                 23630 
                 31016519 
                 5′ (*)-GTAATGTTTGAGTTTTGAGTATTTTC 3′ 
               
               
                   
               
               
                 MLH1-9A-3-as 
                 70 
                 23834 
                 37016723 
                 23853 
                 37016742 
                 5′ CAGAAATTTTTCCATGGTCC 3′ 
               
               
                   
               
               
                 MLH1-9B-s 
                 71 
                 23554 
                 37016443 
                 23575 
                 37016464 
                 5′ (*)-CAAAGTTAGTTTATGGGAAGGA 3′ 
               
               
                   
               
               
                 MLH1-9B-as 
                 72 
                 23741 
                 37016630 
                 23764 
                 31016653 
                 5′ GAAGAGTAAGAAGATGCACTTCTT 3′ 
               
               
                   
               
               
                 MLH1-9C-s 
                 73 
                 23698 
                 37016587 
                 23720 
                 37016609 
                 5′ (*)-CTTCAAAATGAATGGTTACATAT 3′ 
               
               
                   
               
               
                 MLH1-9C-as 
                 74 
                 23810 
                 37016699 
                 23827 
                 37016716 
                 5′ ATTCCCTGTGGGTGTTTC 3′ 
               
               
                   
               
               
                 exon 10 
               
               
                 MLH1-10-s: 
                 78 
                 26665 
                 37019554 
                 26682 
                 37019571 
                 5′ (*)-TGAATGTACACCTGTGAC 3′ 
               
               
                   
               
               
                 MLH1-10-as: 
                 79 
                 26861 
                 37019750 
                 26878 
                 37019767 
                 5′ TAGAACATCTGTTCCTTG 3′ 
               
               
                   
               
               
                 exon 11 
               
               
                 MLH1-11A-s: 
                 83 
                 29423 
                 37022312 
                 29439 
                 37022328 
                 5′ (*)-TTGACCACTGTGTCATC 3′ 
               
               
                   
               
               
                 MLH1-11A-as: 
                 84 
                 25951 
                 37018840 
                 29606 
                 37022495 
                 5′ GTGCAGGAAGTGAACT 3′ 
               
               
                   
               
               
                 MLH1-11B-s: 
                 85 
                 29553 
                 37022442 
                 29571 
                 37022460 
                 5′ (*)-CAGAATGTGGATGTTAATG 3′ 
               
               
                   
               
               
                 MLH1-11B-as: 
                 86 
                 29658 
                 37022547 
                 29672 
                 37022561 
                 5′ GGAGGAATTGGAGCC 3′ 
               
               
                   
               
               
                 MLH1-11C-s4: 
                 87 
                 29631 
                 37022520 
                 29647 
                 37022536 
                 5′ CAGCAGCACATCGAGAG 3′ 
               
               
                   
               
               
                 MLH1-11C-as4: 
                 88 
                 29746 
                 37022635 
                 29763 
                 37022652 
                 5′ (*)-ATCTGGGCTCTCACGTCT 3′ 
               
               
                   
               
               
                 exon 12 
               
               
                 MLH1-12B-s: 
                 92 
                 34849 
                 37027738 
                 34869 
                 37027758 
                 5′ (*)-TTTTTTTTAATACAGACTTTG 3′ 
               
               
                   
               
               
                 MLH1-12B-as: 
                 93 
                 35049 
                 37027938 
                 35063 
                 37027952 
                 5′ GTGACAATGGCCTGG 3′ 
               
               
                   
               
               
                 MLH1-12C-s: 
                 94 
                 35009 
                 37027898 
                 35024 
                 37027913 
                 5′ CATTTCTGCAGCCTCT 3′ 
               
               
                   
               
               
                 MLH1-12C-as: 
                 95 
                 35142 
                 37028031 
                 35156 
                 37028045 
                 5′ (*)-TTTTTGGCAGCCACT 3′ 
               
               
                   
               
               
                 MLH1-12D-s3: 
                 96 
                 35130 
                 37028019 
                 35145 
                 37028034 
                 5′ AGCCCCTGCTGAAGTG 3′ 
               
               
                   
               
               
                 MLH1-12D-as3: 
                 97 
                 35274 
                 37028163 
                 35294 
                 37028183 
                 5′ (*)-AGAAGGCAGTTTTATTACAGA 3′ 
               
               
                   
               
               
                 MLH1-12E-s: 
                 98 
                 35036 
                 37027925 
                 35051 
                 37027940 
                 5′ (*)-TGTCCAGTCAGCCCCA 3′ 
               
               
                   
               
               
                 MLH1-12E-as: 
                 99 
                 35146 
                 37028035 
                 35162 
                 37028051 
                 5′ CTCTGATTTTTGGCAGC 3′ 
               
               
                   
               
               
                 exon 13 
               
               
                 MLH1-13A-s: 
                 106 
                 37950 
                 37030839 
                 37966 
                 37030855 
                 5′ (*)-AATTTGGCTAAGTTTAA 3′ 
               
               
                   
               
               
                 MLH1-13A-as: 
                 107 
                 37950 
                 37030839 
                 37966 
                 37030855 
                 5′ GGAATCATCTTCCACC 3′ 
               
               
                   
               
               
                 MLH1-13B-s2: 
                 108 
                 38003 
                 37030892 
                 38021 
                 37030910 
                 5′ (*)-CATTGCAGAAAGAGACATC 3′ 
               
               
                   
               
               
                 MLH1-13B-as3: 
                 109 
                 38093 
                 37030982 
                 38112 
                 37031001 
                 5′ CGCCCGCCGCGGTGAGGTTAATGATCCTTCT 3′ 
               
               
                   
               
               
                 MLH1-13C-s1: 
                 110 
                 38053 
                 37030942 
                 38073 
                 37030962 
                 5′ (*)-TGATTCCCGAAAGGAAATGAC 3′ 
               
               
                   
               
               
                 MLH1-13C-as1: 
                 111 
                 38153 
                 37031042 
                 38180 
                 37031069 
                 5′ CAGGCCACAGCGTTTACGTACCCTCATG 3′ 
               
               
                   
               
               
                 MLH1-13D-s: 
                 112 
                 38102 
                 37030991 
                 38122 
                 37031011 
                 5′ (*)-ATTAACCTCACTAGTGTTTTG 3′ 
               
               
                   
               
               
                 MLH1-13D-as: 
                 113 
                 38186 
                 37031075 
                 38201 
                 37031090 
                 5′ TGAGGCCCTATGCATC 3′ 
               
               
                   
               
               
                 exon 14 
               
               
                 MLH1-14A-s: 
                 117 
                 49344 
                 37042233 
                 49359 
                 37042248 
                 5′ (*)-GGTCAATGAAGTGGGG 3′ 
               
               
                   
               
               
                 MLH1-14A-as: 
                 118 
                 49432 
                 37042321 
                 49448 
                 37042337 
                 5′ CCACGAAGGAGTGGTTA 3′ 
               
               
                   
               
               
                 MLH1-14B-s: 
                 119 
                 49411 
                 37042300 
                 49426 
                 37042315 
                 5′ AGTTCTCCGGGAGATG 3′ 
               
               
                   
               
               
                 MLH1-14B-as: 
                 120 
                 49596 
                 37042485 
                 49612 
                 37042501 
                 5′ (*)-TACCTCATGCTGCTCTC 3′ 
               
               
                   
               
               
                 exon 15 
               
               
                 MLH1-15-s: 
                 124 
                 51403 
                 37044292 
                 51419 
                 37044308 
                 5′ TTCAGGGATTACTTCTC 3′ 
               
               
                   
               
               
                 MLH1-15-as: 
                 125 
                 51637 
                 37044526 
                 51656 
                 37044545 
                 5′ (*)-GAAAAATTTAACATACTACA 3′ 
               
               
                   
               
               
                 exon 16 
               
               
                 MLH1-16A-s: 
                 129 
                 56658 
                 37049547 
                 56674 
                 37049563 
                 5′ (*)-GCCATTCTGATAGTGGA 3′ 
               
               
                   
               
               
                 MLH1-16A-as2: 
                 130 
                 56768 
                 37049657 
                 56786 
                 37049675 
                 5′ TCTAAGGCAAGCATGGCAA 3′ 
               
               
                   
               
               
                 MLH1-16B-s: 
                 131 
                 56752 
                 37049641 
                 56765 
                 37049654 
                 5′ GCACCGCTCTTTGA 3′ 
               
               
                   
               
               
                 MLH1-16B-as: 
                 132 
                 56914 
                 37049803 
                 56930 
                 37049819 
                 5′ (*)-GTATAAGAATGGCTGTCA 3′ 
               
               
                   
               
               
                 MLH1-16C-s2: 
                 133 
                 56868 
                 37049757 
                 56884 
                 37049773 
                 5′ GGCTGAGATGCTTGCAG 3′ 
               
               
                   
               
               
                 MLH1-16C-as2: 
                 134 
                 56967 
                 37049856 
                 56981 
                 37049870 
                 5′ (*)-CATGAGCCACCGCAC 3′ 
               
               
                   
               
               
                 exon 17 
               
               
                 MLH1-17-s: 
                 138 
                 57689 
                 37050578 
                 57706 
                 37050595 
                 5′ (*)-TGTTTAAACTATGACAGCA 3′ 
               
               
                   
               
               
                 MLH1-17-as: 
                 139 
                 57892 
                 37050781 
                 57906 
                 37050795 
                 5′ TGGTCATTTGCCCTT 3′ 
               
               
                   
               
               
                 exon 18 
               
               
                 MLH1-18A-s: 
                 143 
                 58060 
                 37050949 
                 58077 
                 37050966 
                 5′ (*)-TGTGATCTCCGTTTAGAA 3′ 
               
               
                   
               
               
                 MLH1-18A-as2: 
                 144 
                 58220 
                 37051109 
                 58236 
                 37051125 
                 5′ CTGAGAGGGTCGACTCC 3′ 
               
               
                   
               
               
                 MLH1-18B-s3: 
                 145 
                 58179 
                 37051068 
                 58197 
                 37051086 
                 5′ (*)-TGCGCTATGTTCTATTCCA 3′ 
               
               
                   
               
               
                 MLH1-18B-as3: 
                 146 
                 58264 
                 37051153 
                 58280 
                 37051169 
                 5′ GCCGCCCCCGCCCGCTAGTCCTGGGGTGCCA 3′ 
               
               
                   
               
               
                 exon 19 
               
               
                 MLH1-19A-s: 
                 150 
                 59615 
                 37052504 
                 59631 
                 37052520 
                 5′ CAAGTCTTTCCAGACCC 3′ 
               
               
                   
               
               
                 MLH1-19A-as: 
                 151 
                 59843 
                 37052732 
                 59860 
                 37052749 
                 5′ (*)-TGTATAGATCAGGCAGGT 3′ 
               
               
                   
               
               
                 MLH1-19B-s4 
                 153 
                 59774 
                 37052663 
                 59790 
                 37052679 
                 5′ AAGCCTTGCGCTCACAC 3 
               
               
                   
               
               
                 MLH1-19B-as4 
                 155 
                 59867 
                 37052756 
                 59891 
                 37052780 
                 5′ (*)-AATAACCATATTTAACACCTCTCAA 3′ 
               
               
                   
               
               
                 MLH1-19C-s: 
                 152 
                 59813 
                 37052702 
                 59833 
                 37052722 
                 5′ (*)-CAGAAGATGGAAATATCCTGC 3′ 
               
               
                   
               
               
                 MLH1-19C-as: 
                 153 
                 59937 
                 37052826 
                 59962 
                 37052851 
                 5′ CCGCCCGTGTATATCACACTTTGATACAACACT3′ 
               
               
                   
               
               
                 * clamp is 
                 344 
                   
                   
                   
                   
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG 
               
               
                   
               
            
           
           
               
            
               
                 MLH1 Sequencing Primers 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 this is 5′-3′ sequence for each sense and 
                   
               
               
                   
                   
                   
                   
                   
                   
                 antisense primer. Primers have tags** 
               
               
                 MLH1-1seq-s 
                 13 
                 2562 
                 36995451 
                 2581 
                 36995470 
                 TCTGCCTTTTTCTTCCATCGGGGCTTCAGGGAGGGACGAAGA 
               
               
                   
               
               
                 MLH1-1seq-as 
                 14 
                 2979 
                 36995868 
                 2998 
                 36995887 
                 TCCCCAACCCCCTAAAGCGATGCGCTGTACATGCCTCTGC 
               
               
                   
               
               
                 MLH1-2seq-s 
                 20 
                 5653 
                 36998542 
                 5672 
                 36998561 
                 TCTGCCTTTTTCTTCCATCGGGTGCCCGTCTCTTCCCTCTCT 
               
               
                   
               
               
                 MLH1-2seq-as 
                 21 
                 6085 
                 36998974 
                 6104 
                 36998993 
                 TCCCCAACCCCCTAAAGCGACCTGAACAGTGCCCAGCAAA 
               
               
                   
               
               
                 MLH1-3seq-s 
                 27 
                 9947 
                 37002836 
                 9970 
                 37002859 
                 TCTGCCTTTTTCTTCCATCGGGCAAGACTCTGTCTCAAAGGAGGTT 
               
               
                   
               
               
                 MLH1-3seq-as2 
                 30 
                 10401 
                 37003290 
                 10425 
                 37003314 
                 TCCCCAACCCCCTAAAGCGACATTAAGTTTGCTCAGATTTGCATA 
               
               
                   
               
               
                 MLH1-4seq-s 
                 36 
                 13474 
                 37006363 
                 13495 
                 37006384 
                 TCTGCCTTTTTCTTCCATCGGGCATGTCATCAAAGCAAGTGAGC 
               
               
                   
               
               
                 MLH1-4seq-as 
                 37 
                 13759 
                 37006648 
                 13782 
                 37006671 
                 TCCCCAACCCCCTAAAGCGATGAGACAGGATTACTCTGAGACCT 
               
               
                   
               
               
                 MLH1-5seq-s2 
                 47 
                 16159 
                 37009048 
                 16182 
                 37009071 
                 TCTGCCTTTTTCTTCCATCGGGCCCTTGGGATTAGTATCTATCTCT 
               
               
                   
               
               
                 MLH1-5seq-as 
                 48 
                 16397 
                 37009286 
                 16418 
                 37009307 
                 TCCCCAACCCCCTAAAGCGAGGACCTCCATTAACTAGTGCAA 
               
               
                   
               
               
                 MLH1-6seq-s 
                 52 
                 17877 
                 37010766 
                 17900 
                 37010789 
                 TCTGCCTTTTTCTTCCATCGGGCTGTTAATGCTGTCTTATCCCTGG 
               
               
                   
               
               
                 MLH1-6seq-as 
                 53 
                 18204 
                 37011093 
                 18226 
                 37011115 
                 TCCCCAACCCCCTAAAGCGACCATCTAGCTCAGCAACTGTTCA 
               
               
                   
               
               
                 MLH1-7seq-s 
                 59 
                 20856 
                 37013745 
                 20875 
                 37013764 
                 TCTGCCTTTTTCTTCCATCGGGTTCCATGAAGTTTCTGCTGG 
               
               
                   
               
               
                 MLH1-7seq-as 
                 58 
                 21151 
                 37014040 
                 21172 
                 37014061 
                 TCCCCAACCCCCTAAAGCGACCTTATCTCCACCAGCAAACTA 
               
               
                   
               
               
                 MLH1-8seq-s 
                 66 
                 21100 
                 37013989 
                 21120 
                 37014009 
                 TCTGCCTTTTTCTTCCATCGGGGGTTTATGGGGGATGGTTTTG 
               
               
                   
               
               
                 MLH1-8seq-as 
                 67 
                 21520 
                 37014409 
                 21543 
                 37014432 
                 TCCCCAACCCCCTAAAGCGACGCCACAGAATCTAGGAGATTACA 
               
               
                   
               
               
                 MLH1-9seq-s 
                 75 
                 23462 
                 37016351 
                 23481 
                 37016370 
                 TCTGCCTTTTTCTTCCATCGGGGGTGGGTGAATGGGTGAACA 
               
               
                   
               
               
                 MLH1-9seq-as 
                 76 
                 23875 
                 37016764 
                 23894 
                 37016783 
                 TCCCCAACCCCCTAAAGCGATTTGCCATGAGGTTTCTCCA 
               
               
                   
               
               
                 MLH1-10seq-s 
                 80 
                 26563 
                 37019452 
                 26581 
                 37019470 
                 TCTGCCTTTTTCTTCCATCGGGGCTGGAAAGTGGCGACAGG 
               
               
                   
               
               
                 MLH1-10seq-as 
                 81 
                 26929 
                 37019818 
                 26949 
                 37019838 
                 TCCCCAACCCCCTAAAGCGAGCCAGTGGTGTATGGGATTCA 
               
               
                   
               
               
                 MLH1-11seq-s 
                 89 
                 29324 
                 37022213 
                 29344 
                 37022233 
                 TCTGCCTTTTTCTTCCATCGGGAGACTGAGGCAAAGAAAGATG 
               
               
                   
               
               
                 MLH1-11seq-as 
                 90 
                 29753 
                 37022642 
                 29771 
                 37022660 
                 TCCCCAACCCCCTAAAGCGAAGGCAAAAATCTGGGCTCT 
               
               
                   
               
               
                 MLH1-12seq-s 
                 100 
                 34696 
                 37027585 
                 34714 
                 37027603 
                 TCTGCCTTTTTCTTCCATCGGGTTTCGGGCAGAATTGCTTC 
               
               
                   
               
               
                 MLH1-12seq-as 
                 101 
                 35312 
                 37028201 
                 35334 
                 37028223 
                 TCCCCAACCCCCTAAAGCGAGCAGAGAGAAGATGCAAGTGATT 
               
               
                 alternate 
               
               
                   
               
               
                 MLH1-12seq-s2 
                 103 
                 34453 
                 37027342 
                 34474 
                 37027363 
                 TCTGCCTTTTTCTTCCATCGGGATAGCTGGTGGTGATGGTTGCG 
               
               
                   
               
               
                 MLH1-12seq-as2 
                 104 
                 35345 
                 37028234 
                 35366 
                 37028255 
                 TCCCCAACCCCCTAAAGCGACCATTCCAGCACCATTCCAGAG 
               
               
                   
               
               
                 MLH1-13seq-s 
                 114 
                 37852 
                 37030741 
                 37872 
                 37030761 
                 TCTGCCTTTTTCTTCCATCGGGACTGATCTTGTTGGCCTTCTG 
               
               
                   
               
               
                 MLH1-13seq-as 
                 115 
                 38233 
                 37031122 
                 38252 
                 37031141 
                 TCCCCAACCCCCTAAAGCGATGGCCACTCTGACAACATGA 
               
               
                   
               
               
                 MLH1-14seq-s 
                 121 
                 49268 
                 37042157 
                 49287 
                 37042176 
                 TCTGCCTTTTTCTTCCATCGGGTGTTCGTTTTCACCAGGAGG 
               
               
                   
               
               
                 MLH1-14seq-as 
                 122 
                 49647 
                 37042536 
                 49668 
                 37042557 
                 TCCCCAACCCCCTAAAGCGATCGAACTTGGATTTGAAACCAC 
               
               
                   
               
               
                 MLH1-15seq-s2 
                 126 
                 51361 
                 37044250 
                 51379 
                 37044268 
                 TCTGCCTTTTTCTTCCATCGGGAGATTCCACAGCCAGGCAG 
               
               
                   
               
               
                 MLH1-15seq-as2 
                 127 
                 51683 
                 37044572 
                 51706 
                 37044595 
                 TCCCCAACCCCCTAAAGCGATACCTCCATATGCAAATCATACAA 
               
               
                   
               
               
                 MLH1-16seq-s 
                 135 
                 56582 
                 37049471 
                 56604 
                 37049493 
                 TCTGCCTTTTTCTTCCATCGGGGGTTTTGTTGTGGATTGTTCAGG 
               
               
                   
               
               
                 MLH1-16seq-as 
                 136 
                 56974 
                 37049863 
                 56993 
                 37049882 
                 TCCCCAACCCCCTAAAGCGATGGGATTACAGCCATGAGCC 
               
               
                   
               
               
                 MLH1-17seq-s 
                 140 
                 57580 
                 37050469 
                 57601 
                 37050490 
                 TCTGCCTTTTTCTTCCATCGGGTTTAAGTGTTTAGGTCTGCCCC 
               
               
                   
               
               
                 MLH1-17seq-as 
                 141 
                 59926 
                 37052815 
                 57948 
                 37050837 
                 TCCCCAACCCCCTAAAGCGAGCTATCCCACCCTTATCATCTTT 
               
               
                   
               
               
                 MLH1-18seq-s 
                 147 
                 57927 
                 37050816 
                 57948 
                 37050837 
                 TCTGCCTTTTTCTTCCATCGGGAAGATGATAAGGGTGGGATAGC 
               
               
                   
               
               
                 MLH1-18seq-as 
                 148 
                 58317 
                 37051206 
                 58336 
                 37051225 
                 TCCCCAACCCCCTAAAGCGACCGAAATTTTAGAGATGGGC 
               
               
                   
               
               
                 MLH1-19seq-s 
                 156 
                 59462 
                 37052351 
                 59482 
                 37052371 
                 TCTGCCTTTTTCTTCCATCGGGGCTATGATCACACCACTGCCC 
               
               
                   
               
               
                 MLH1-19seq-as 
                 157 
                 60108 
                 37052997 
                 60129 
                 37053018 
                 TCCCCAACCCCCTAAAGCGACCTCTTTTTGGCATCTGAACTG 
               
               
                   
               
               
                 MSH2/MLH1 
                   
                   
                   
                   
                   
                 tagged primer in DTCS reaction 
               
               
                 ** sense tag is 
                 289 
                   
                   
                   
                   
                 TCTGCCTTTTTCTTCCATCGGG 
               
               
                   
               
               
                 ** antisense tag is 
                 290 
                   
                   
                   
                   
                 TCCCCAACCCCCTAAAGCGA 
               
            
           
           
               
               
               
            
               
                 MLH1 
                 Sequencing Primers internal instead of tagged primer in sense direction 
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 MLH1-3seq-s2-int 
                 29 
                 10094 
                 37002983 
                 10117 
                 37003006 
                 CCTGGATTAAATCAAGAAAATGGG 
                   
               
               
                   
               
               
                 MLH1-12seq-s2-int 
                 102 
                 34861 
                 37027750 
                 34884 
                 37027773 
                 CAGACTTTGCTACCAGGACTTGCT 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE C 
               
               
                   
               
             
            
               
                 Primer master set up for MLH1 and MSH2 
               
            
           
           
               
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 rev. 040404 
               
            
           
           
               
            
               
                 HNPCC ASSAY PCR SET UP AND STACKING AK 040404 
               
            
           
           
               
            
               
                 PART 1: PCR 
               
               
                 Primer plate 
               
               
                 Primer plate has 13.5 ul of primer mix at 5 uM or 10 uM as shown below. Heat sealed. 
               
               
                 Take from freezer, thaw at room temp for a few min, spin down 1 min 1500 g, open carefully. Keep cool on cooler block. 
               
               
                 Log date of primer plate made: 
               
               
                 Log date of primer plate used: 
               
               
                 Log number of primer plate used: 
               
            
           
           
               
               
            
               
                 Visually Inspect volume 
                 ok? 
               
               
                   
               
            
           
           
               
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Run HNPCC PCR program on Biomek. Biomek set up as below. Use fresh box of P20 and P250 in each block. 
               
               
                 7 plate set-up run: 
               
               
                 Pipette manually 67.5 ul of hotstart master mix to each primer well. Pipette up and down three times. Avoid bubbles. 
               
               
                 Pause after transfer for visual inspection and quick spin 1500 g, 1 min if necessary. Make sure all bubbles are gone. 
               
               
                 Place primer plate on robot with other labeled plates. 
               
               
                 Run program, transfer 9 ul of primer/MM from one well to each well of column A-H of corresponding primer plate. 
               
               
                 Multi eject, no tip touch if have P250 (takes up 9 × 9 ul). Asp. height and rate 5/3, eject 10/4. 
               
               
                 Program pauses after all primers have been dispensed. Inspect and quick spin if necessary-otherwise continue. 
               
               
                 Replace primer master with Falcon plate with gDNA in B3 
               
               
                 Add gDNA/water from Falcon rows 1 and 2 with multi20, 6 ul per well, tip touch. 
               
               
                 Asp. Heights and rates are 10/6, 60/3, last 5/6, eject 60/3. Tip change after plate. 
               
               
                 Remove PCR plates from Biomek. 
               
               
                 Carefully shake DNA down from edge of PCR plates, heat seal. Vortex gently 30 sec., spin 1500 g 1 min. 
               
               
                 Run PCR 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 Store plates at −20 unless proceeding to force het and stacking programs. Quick spin prior to storage. 
               
               
                 PART 2: 
               
               
                 Force heteromers: 
               
               
                 Before stacking, force het for 5 min at 95 C., 10 min at 50 C., 4 min/hold. Keep plates at 4. 
               
               
                 PART 3: 
               
               
                 Stacking program 
               
               
                 Take PCR plates from 4 C., fresh Falcon plates and set up Biomek as below. 
               
               
                 Spin PCR plates briefly 1500 g, 1 min. to collect volume. 
               
               
                 Load 200 ul 2x loading dye into rows 1 and 2 of master Falcon plate at A2. 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 continue stacking program 
               
               
                 Transfer is: A2 dye from row 1 to all wells of B2, varied volumes, no tip touch. MP20 (6-13.5 ul) 
               
               
                 A2 dye from rows 2 and 3 to all of B3, varied volumes, no tip touch. MP20 
               
               
                 Asp. Heights and rates are 8/4 and 10/4. 
               
               
                 Tip change after B2 load and after B3 load. Pause. 
               
               
                 PCR product from all plates to B2 or B3 in groups (each sample 4-6 ul; 2-4 samples per group) 
               
               
                 Asp. 3/4 and eject 5/5 blowout 
               
               
                 Seal plates with clear plastic and store at 4 C. Store loading plates at 4 C. for gel loading. 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE D 
               
               
                   
               
             
            
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE E 
               
               
                   
               
             
            
               
                 MSH2 and MLH1 SEQ Primers 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Exon 
                 MSH2 Primer set for PCR 
                 PCR anneal 
               
               
                   
               
               
                 1 
                 first choice: MSH2-1seq-s2/as2 
                 61.8 
               
               
                 1 
                 second choice: MSH2-1seq-s/as 
                 61.8 
               
               
                 2 
                 first choice MSH2-2seq-s2/as2 
                 61.8 
               
               
                 2 
                 second choice: MSH2-2seq-s3/as3 
                 69 
               
               
                 3 
                 first choice: MSH2-3seq-s/as4 
                 61.8 
               
               
                 3 
                 second choice: MSH2-3seq-s4/as4 
                 56.7 
               
               
                 4 
                 MSH2-4seq-s2/as2 
                 61.8 
               
               
                 5 
                 first choice: MSH2-5seq-s3/as3 
                 61.8 
               
               
                 5 
                 second choice: MSH2-5seq-s2/as2 
                 61.8 
               
               
                 6 
                 MSH2-6seq-s/as 
                 56.7 
               
               
                 7 
                 MSH2-7seq-s3/as2 
                 61.8 
               
               
                 8 
                 MSH2-8seq-s/as 
                 56.7 
               
               
                 9 
                 MSH2-9seq-s/as 
                 56.7 
               
               
                 10 
                 MSH2-10seq-s/as 
                 56.7 
               
               
                 11 
                 MSH2-11seq-s/as 
                 56.7 
               
               
                 12 
                 MSH2-12seq-s/as 
                 61.8 
               
               
                 13 
                 MSH2-13seq-s/as 
                 56.7 
               
               
                 (GAP) = 14 
                 (MSH2-GAPseq-s/as) = (MSH2-14seq-s/as) 
                 61.8 
               
               
                 (14) = 15 
                 (MSH2-14seq-s/as) = (MSH2-15seq-s/as) 
                 56.7 
               
               
                 (15) = 16 
                 (MSH2-15seq-s/as) = (MSH2-16seq-s/as) 
                 56.7 
               
               
                   
               
               
                 Exons 
                 MSH2 Sequencing Primers 
               
               
                   
               
               
                 all exons 
                 MSH2 s tag 
               
               
                 all exons 
                 MSH2 as tag 
               
               
                 2 
                 MSH2-2seq-s2-int added 081303 
               
               
                 5 
                 MSH2-5seq-as2-int added 081303 
               
               
                   
               
               
                 Exon 
                 MLH1 Primer set for PCR. 
                 PCR anneal 
               
               
                   
               
               
                 1 
                 MLH1-1seq-s/as 
                 63.4 
               
               
                 2 
                 MLH1-2seq-s/as 
                 63.4 
               
               
                 3 
                 MLH1-3seq-s/as2 
                 63.4 
               
               
                 4 
                 MLH1-4seq-s/as 
                 63.4 
               
               
                 5 
                 MLH1-5seq-s2/as1 
                 59.6 
               
               
                 6 
                 MLH1-6seq-s/as 
                 63.4 
               
               
                 7 
                 MLH1-7seq-s/as 
                 59.6 
               
               
                 8 
                 MLH1-8seq-s/as 
                 59.6 
               
               
                 9 
                 MLH1-9seq-s/as 
                 63.4 
               
               
                 10 
                 MLH1-10seq-s/as 
                 63.4 
               
               
                 11 
                 MLH1-11seq-s/as 
                 63.4 
               
               
                 12 
                 first choice: MLH1-12seq-s/as 
                 59.6 
               
               
                 12 
                 second choice: MLH1-12seq-s2/as2 
                 59.6 
               
               
                 13 
                 MLH1-13seq-s/as 
                 63.4 
               
               
                 14 
                 MLH1-14seq-s/as 
                 63.4 
               
               
                 15 
                 MLH1-15seq-s2/as2 
                 63.4 
               
               
                 16 
                 MLH1-16seq-s/as 
                 63.4 
               
               
                 17 
                 MLH1-17seq-s/as 
                 63.4 
               
               
                 18 
                 MLH1-18seq-s/as 
                 63.4 
               
               
                 19 
                 MLH1-19seq-s/as 
                 63.4 
               
               
                   
               
               
                 Exons 
                 MLH1 Sequencing Primers 
               
               
                   
               
               
                 all but 3, 12 
                 MSH2 s tag 
               
               
                 all 
                 MSH2 as tag 
               
               
                 3 
                 MLH1-3seq-s2-int 
               
               
                 12 
                 MLH1-12seq-s2-int 
               
               
                   
               
            
           
           
               
            
               
                 PCR Volumes 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Add 
                 5 
                 ul 
                 TaqMM or Hotstar TaqMM 
                   
               
               
                   
                   
                 0.5 
                 ul 
                 gDNA 
               
               
                   
                   
                 1.0 
                 ul 
                 primer mix at 5 uM S and AS primer 
               
               
                   
                   
                 3.5 
                 ul 
                 water 
               
               
                   
                   
                 10 
                 ul 
                 total 
               
            
           
           
               
            
               
                 PCR Conditions 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 1 
                 95 C. 
                 minutes 
                 or 15 min with hotstar 
                   
               
               
                   
                 2 
                 94 C. 
                 30 seconds 
                 TAQMM 
               
               
                   
                 3 
                 annealing temp as indicated above 
                 30 seconds 
               
               
                   
                 4 
                 72 C. 
                 45-60 seconds 
                 4 links to 2 30-35x 
               
               
                   
                 5 
                 72 C. 
                 10 minutes 
               
               
                   
                 6 
                  4 C. 
                 forever 
               
            
           
           
               
            
               
                 EXO SAP IT Volumes 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 Exo (uL) 
                   
                 PCR Prod (uL) 
                   
               
               
                   
                 Add 
                 1 
                   
                 to 2.5 
               
               
                   
                   
                 2 
                   
                 5 
               
            
           
           
               
            
               
                 EXO SAP IT Conditions 
               
            
           
           
               
               
               
               
               
            
               
                   
                 1 
                 37 C. 
                 60 minutes 
                   
               
               
                   
                 2 
                 72 C. 
                 15 minutes 
               
            
           
           
               
            
               
                 DTCS Volumes 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Add 
                 4.0-4.5 
                 uL 
                 of dH2O 
                   
               
               
                   
                   
                 0.5-1.0 
                 uL 
                 of Exo Sap it Product 
               
               
                   
                   
                 1.0 
                 uL 
                 of 1.6 uM Primer (sense or anti-sense) 
               
               
                   
                   
                 4.0 
                 uL 
                 DTCS solution 
               
               
                   
                   
                 10 
                 uL 
                 Total 
               
            
           
           
               
            
               
                 DTCS Conditions 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 1 
                 96 C. 
                 20 seconds 
                   
                   
               
               
                   
                 2 
                 50 C. 
                 20 seconds 
               
               
                   
                 3 
                 60 C. 
                 4 minutes 
                 3 links to 1 35x 
               
               
                   
                 4 
                  4 C. 
                 forever 
               
            
           
           
               
            
               
                 Primer stock 5 uM mixed: 
               
            
           
           
               
               
               
               
            
               
                 10 
                 ul 
                 50 uM sense primer 
                   
               
               
                 10 
                 ul 
                 50 uM antisense primer 
               
               
                 80 
                 ul 
                 water 
               
               
                 100 
                 ul 
                 total 
               
            
           
           
               
            
               
                 CEQ 2000 Run Conditions 
               
               
                 Injection Time: 20 seconds 
               
               
                 Run Time: 65-85 minutes 
               
               
                 these times are exceptions to the default parameters 
               
               
                 Rev 002 MSH2 and MLH1 Sequencing Primers 
               
               
                 AK 
               
               
                 4/25/2003 
               
               
                 8/15/2003 
               
               
                 9/17/2003 
               
               
                 2/13/2004 
               
               
                 3/6/2004 
               
               
                 3/26/2004 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Exon 
                 Primer 
                 Seq. ID No. 
                 Sequence 
               
               
                   
               
            
           
           
               
            
               
                 MSH2 and MLH1 Sequencing Primers 
               
               
                 2/13/2004 
               
               
                 AK 
               
               
                 MSH2 
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 MSH2-1seq-s2 
                 162 
                 TCTGCCTTTTTCTTCCATCGGGGCGCAGTAGCTAAAGTCACCAG 
                   
               
               
                   
                 MSH2-1seq-as2 
                 163 
                 TCCCCAACCCCCTAAAGCGAGAATCCGCACAAGCACCAAC 
               
               
                   
               
               
                   
                 alternate 
               
               
                 1 
                 MSH2-1seq-s 
                 160 
                 TCTGCCTTTTTCTTCCATCGGGGGCGGGAAACAGCTTAGTGG 
               
               
                   
                 MSH2-1seq-as 
                 161 
                 TCCCCAACCCCCTAAAGCGACGCACTGGAGAGGCTGCTCA 
               
               
                   
               
               
                 2 
                 MSH2-2seq-s2 
                 169 
                 TCTGCCTTTTTCTTCCATCGGGTGCTGCCATCCATGTAAGAC 
               
               
                   
                 MSH2-2seq-as2 
                 170 
                 TCCCCAACCCCCTAAAGCGACCAGCCAAACTGCAACTTTT 
               
               
                   
               
               
                   
                 alternate 
               
               
                 2 
                 MSH2-2seq-s3 
                 171 
                 TCTGCCTTTTTCTTCCATCGGGTTCCTCCTTGCCTTCTGCCAT 
               
               
                   
                 MSH2-2seq-as3 
                 172 
                 TCCCCAACCCCCTAAAGCGAGGGATTACAAGCATGAGCCACTG 
               
               
                   
               
               
                 3 
                 MSH2-3seq-s 
                 291 
                 TCTGCCTTTTTCTTCCATCGGGCAGAGCAAGACTTCATCTCA 
               
               
                   
                 MSH2-3seq-as4 
                 181 
                 TCCCCAACCCCCTAAAGCGACCTTAAATGAAACAGTATCATGTCAA 
               
               
                   
               
               
                   
                 alternate 
               
               
                   
                 MSH2-3seq-s4 
                 180 
                 TCTGCCTTTTTCTTCCATCGGGGGTTCATAGAGTTTGGAATTTTTCC 
               
               
                   
                 MSH2-3seq-as4 
                 181 
                 TCCCCAACCCCCTAAAGCGACCTTAAATGAAACAGTATCATGTCAA 
               
               
                   
               
               
                 4 
                 MSH2-4seq-s2 
                 191 
                 TCTGCCTTTTTCTTCCATCGGGGCATTCCATCCTGGGCGA 
               
               
                   
                 MSH2-4seq-as2 
                 192 
                 TCCCCAACCCCCTAAAGCGACAGCCTGGGCAACAAAAGTG 
               
               
                   
               
               
                 5 
                 MSH2-5seq-s3 
                 200 
                 TCTGCCTTTTTCTTCCATCGGGAGTTTTGATGGACATTTGGGTAA 
               
               
                   
                 MSH2-5seq-as3 
                 201 
                 TCCCCAACCCCCTAAAGCGAGTTAAAAAGTGGAGTGGAGGAGG 
               
               
                   
               
               
                   
                 alternate 
               
               
                 5 
                 MSH2-5seq-s2 
                 198 
                 TCTGCCTTTTTCTTCCATCGGGTTCTTGGTTTGGATTGGGAAGG 
               
               
                   
                 MSH2-5seq-as2 
                 199 
                 TCCCCAACCCCCTAAAGCGAGGGGAGAGAAAAATACAGCCAT 
               
               
                   
               
               
                 6 
                 MSH2-6seq-s 
                 209 
                 TCTGCCTTTTTCTTCCATCGGGTGAACATACGGATTAAGAGG 
               
               
                   
                 MSH2-6seq-as 
                 210 
                 TCCCCAACCCCCTAAAGCGACATATACTTCCAAAACTGCA 
               
               
                   
               
               
                 7 
                 MSH2-7seq-s3 
                 218 
                 TCTGCCTTTTTCTTCCATCGGGGCTGATTTAGTTGAGACTTACGTGC 
               
               
                   
                 MSH2-7seq-as2 
                 219 
                 TCCCCAACCCCCTAAAGCGAGAGGACAGCACATTGCCAAG 
               
               
                   
               
               
                 8 
                 MSH2-8seq-s 
                 229 
                 TCTGCCTTTTTCTTCCATCGGGAATAGTAACTTTGGAGACCTGC 
               
               
                   
                 MSH2-8seq-as 
                 230 
                 TCCCCAACCCCCTAAAGCGACAGGACAGTTATGCCCAATA 
               
               
                   
               
               
                 9 
                 MSH2-9seq-s 
                 234 
                 TCTGCCTTTTTCTTCCATCGGGGAAAGTCCTTAATAGTTGTGACTG 
               
               
                   
                 MSH2-9seq-as 
                 235 
                 TCCCCAACCCCCTAAAGCGAGGGAACTTATAAAATAATTCTGGC 
               
               
                   
               
               
                 10 
                 MSH2-10seq-s 
                 243 
                 TCTGCCTTTTTCTTCCATCGGGTCATAAGGGAGTTAAGGATTT 
               
               
                   
                 MSH2-10seq-as 
                 244 
                 TCCCCAACCCCCTAAAGCGACTGCTCTATGGAAGAAAGCT 
               
               
                   
               
               
                 11 
                 MSH2-11seq-s 
                 250 
                 TCTGCCTTTTTCTTCCATCGGGCATTTGTCCCTAAGGAGTTGTTC 
               
               
                   
                 MSH2-11seq-as 
                 251 
                 TCCCCAACCCCCTAAAGCGACAGAATGTAATGGCTTGCGA 
               
               
                   
               
               
                 12 
                 MSH2-12seq-s 
                 261 
                 TCTGCCTTTTTCTTCCATCGGGTGTTGAGTTTTAGGTGGGTTCC 
               
               
                   
                 MSH2-12seq-as 
                 262 
                 TCCCCAACCCCCTAAAGCGATACCCCCACAAAGCCCAAA 
               
               
                   
               
               
                 13 
                 MSH2-13seq-s 
                 270 
                 TCTGCCTTTTTCTTCCATCGGGGCTATGTCAGTGTAAACCTACGC 
               
               
                   
                 MSH2-13seq-as 
                 271 
                 TCCCCAACCCCCTAAAGCGACTTCTCACAGGACAGAGACATACA 
               
               
                   
               
               
                 (GAP) = ex14 
                 (MSH2-GAPseq-s) = MSH2-14seq-s 
                 277 
                 TCTGCCTTTTTCTTCCATCGGGATGTTTGTGGCATATCCTTCC 
               
               
                   
                 (MSH2-GAPseq-as) = MSH2-14seq- 
                 278 
                 TCCCCAACCCCCTAAAGCGATAGTAAGTTTCCCATTACCAAGTTC 
               
               
                   
                 as 
               
               
                   
               
               
                 (14) = ex15 
                 (MSH2-14seq-s) = MSH2-15seq-s 
                 282 
                 TCTGCCTTTTTCTTCCATCGGGTTGGCTAATGTATTTGAAGTAATCC 
               
               
                   
                 (MSH2-14seq-as) = MSH2-15seq-as 
                 283 
                 TCCCCAACCCCCTAAAGCGAACACAGAGGAAAACAAACAACAA 
               
               
                   
               
               
                 (15) = ex16 
                 (MSH2-15seq-s) = MSH2-16seq-s 
                 287 
                 TCTGCCTTTTTCTTCCATCGGGGTAAAGGTGGAGGAATTTGGG 
               
               
                   
                 (MSH2-15seq-as) = MSH2-16seq-as 
                 288 
                 TCCCCAACCCCCTAAAGCGAGGCACTGACAGTTAACACTATGGA 
               
               
                   
               
            
           
           
               
            
               
                 MLH1 
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 MLH1-1seq-s 
                 13 
                 TCTGCCTTTTTCTTCCATCGGGGCTTCAGGGAGGGACGAAGA 
                   
               
               
                   
                 MLH1-1seq-as 
                 14 
                 TCCCCAACCCCCTAAAGCGATGCGCTGTACATGCCTCTGC 
               
               
                   
               
               
                 2 
                 MLH1-2seq-s 
                 20 
                 TCTGCCTTTTTCTTCCATCGGGTGCCCGTCTCTTCCCTCTCT 
               
               
                   
                 MLH1-2seq-as 
                 21 
                 TCCCCAACCCCCTAAAGCGACCTGAACAGTGCCCAGCAAA 
               
               
                   
               
               
                 3 
                 MLH1-3seq-s 
                 27 
                 TCTGCCTTTTTCTTCCATCGGGCAAGACTCTGTCTCAAAGGAGGTT 
               
               
                   
                 MLH1-3seq-as2 
                 30 
                 TCCCCAACCCCCTAAAGCGACATTAAGTTTGCTCAGATTTGCATA 
               
               
                   
               
               
                 4 
                 MLH1-4seq-s 
                 36 
                 TCTGCCTTTTTCTTCCATCGGGCATGTCATCAAAGCAAGTGAGC 
               
               
                   
                 MLH1-4seq-as 
                 37 
                 TCCCCAACCCCCTAAAGCGATGAGACAGGATTACTCTGAGACCT 
               
               
                   
               
               
                 5 
                 MLH1-5seq-s2 
                 47 
                 TCTGCCTTTTTCTTCCATCGGGCCCTTGGGATTAGTATCTATCTCT 
               
               
                   
                 MLH1-5seq-as 
                 48 
                 TCCCCAACCCCCTAAAGCGAGGACCTCCATTAACTAGTGCAA 
               
               
                   
               
               
                 6 
                 MLH1-6seq-s 
                 52 
                 TCTGCCTTTTTCTTCCATCGGGCTGTTAATGCTGTCTTATCCCTGG 
               
               
                   
                 MLH1-6seq-as 
                 53 
                 TCCCCAACCCCCTAAAGCGACCATCTAGCTCAGCAACTGTTCA 
               
               
                   
               
               
                 7 
                 MLH1-7seq-s 
                 57 
                 TCTGCCTTTTTCTTCCATCGGGTTCCATGAAAGTTTCTGCTGG 
               
               
                   
                 MLH1-7seq-as 
                 58 
                 TCCCCAACCCCCTAAAGCGACCTTATCTCCACCAGCAAACTA 
               
               
                   
               
               
                 8 
                 MLH1-8seq-s 
                 66 
                 TCTGCCTTTTTCTTCCATCGGGGGTTTATGGGGGATGGTTTTG 
               
               
                   
                 MLH1-8seq-as 
                 67 
                 TCCCCAACCCCCTAAAGCGACGCCACAGAATCTAGGAGATTACA 
               
               
                   
               
               
                 9 
                 MLH1-9seq-s 
                 75 
                 TCTGCCTTTTTCTTCCATCGGGGGTGGGTGAATGGGTGAACA 
               
               
                   
                 MLH1-9seq-as 
                 76 
                 TCCCCAACCCCCTAAAGCGATTTGCCATGAGGTTTCTCCA 
               
               
                   
               
               
                 10 
                 MLH1-10seq-s 
                 80 
                 TCTGCCTTTTTCTTCCATCGGGGCTGGAAAGTGGCGACAGG 
               
               
                   
                 MLH1-10seq-as 
                 81 
                 TCCCCAACCCCCTAAAGCGAGCCAGTGGTGTATGGGATTCA 
               
               
                   
               
               
                 11 
                 MLH1-11seq-s 
                 89 
                 TCTGCCTTTTTCTTCCATCGGGAGACTGAGGCAAAGAAAGATG 
               
               
                   
                 MLH1-11seq-as 
                 90 
                 TCCCCAACCCCCTAAAGCGAAGGCAAAAATCTGGGCTCT 
               
               
                   
               
               
                 12 
                 MLH1-12seq-s 
                 100 
                 TCTGCCTTTTTCTTCCATCGGGTTTCGGGCAGAATTGCTTC 
               
               
                   
                 MLH1-12seq-as 
                 101 
                 TCCCCAACCCCCTAAAGCGAGCAGAGAGAAGATGCAAGTGATT 
               
               
                   
                 alternate 
               
               
                   
               
               
                 12 
                 MLH1-12seq-s2 
                 103 
                 TCTGCCTTTTTCTTCCATCGGGATAGCTGGTGGTGATGGTTGCG 
               
               
                   
                 MLH1-12seq-as2 
                 104 
                 TCCCCAACCCCCTAAAGCGACCATTCCAGCACCATTCCAGAG 
               
               
                   
               
               
                 13 
                 MLH1-13seq-s 
                 114 
                 TCTGCCTTTTTCTTCCATCGGGACTGATCTTGTTGGCCTTCTG 
               
               
                   
                 MLH1-13seq-as 
                 115 
                 TCCCCAACCCCCTAAAGCGATGGCCACTCTGACAACATGA 
               
               
                   
               
               
                 14 
                 MLH1-14seq-s 
                 121 
                 TCTGCCTTTTTCTTCCATCGGGTGTTCGTTTTCACCAGGAGG 
               
               
                   
                 MLH1-14seq-as 
                 122 
                 TCCCCAACCCCCTAAAGCGATCGAACTTGGATTTGAAACCAC 
               
               
                   
               
               
                 15 
                 MLH1-15seq-s2 
                 126 
                 TCTGCCTTTTTCTTCCATCGGGAGATTCCACAGCCAGGCAG 
               
               
                   
                 MLH1-15seq-as2 
                 127 
                 TCCCCAACCCCCTAAAGCGATACCTCCATATGCAAATCATACAA 
               
               
                   
               
               
                 16 
                 MLH1-16seq-s 
                 135 
                 TCTGCCTTTTTCTTCCATCGGGGGTTTTGTTGTGGATTGTTCAGG 
               
               
                   
                 MLH1-16seq-as 
                 136 
                 TCCCCAACCCCCTAAAGCGATGGGATTACAGCCATGAGCC 
               
               
                   
               
               
                 17 
                 MLH1-17seq-s 
                 140 
                 TCTGCCTTTTTCTTCCATCGGGTTTAAGTGTTTAGGTCTGCCCC 
               
               
                   
                 MLH1-17seq-as 
                 141 
                 TCCCCAACCCCCTAAAGCGAGCTATCCCACCCTTATCATCTTT 
               
               
                   
               
               
                 18 
                 MLH1-18seq-s 
                 147 
                 TCTGCCTTTTTCTTCCATCGGGAAGATGATAAGGGTGGGATAGC 
               
               
                   
                 MLH1-18seq-as 
                 148 
                 TCCCCAACCCCCTAAAGCGACCGAAATTTTAGAGATGGGC 
               
               
                   
               
               
                 19 
                 MLH1-19seq-s 
                 156 
                 TCTGCCTTTTTCTTCCATCGGGGCTATGATCACACCACTGCCC 
               
               
                   
                 MLH1-19seq-as 
                 157 
                 TCCCCAACCCCCTAAAGCGACCTCTTTTTGGCATCTGAACTG 
               
               
                   
               
            
           
           
               
               
               
            
               
                 MSH2/MLH1 
                 Sequencing Primers 
                   
               
            
           
           
               
               
               
               
               
            
               
                 all exons 
                 MSH2 s tag 
                 289 
                 TCTGCCTTTTTCTTCCATCGGG 
                   
               
               
                   
               
               
                 all exons 
                 MSH2 as tag 
                 290 
                 TCCCCAACCCCCTAAAGCGA 
               
               
                   
               
            
           
           
               
               
               
            
               
                 MLH1 
                 Sequencing Primers internal instead of tagged primer in sense direction 
                   
               
            
           
           
               
               
               
               
               
            
               
                 3 
                 MLH1-3seq-s2-int 
                 29 
                 ctggattaaatcaagaaaatggg 
                   
               
               
                   
               
               
                 12 
                 MLH1-12seq-s2-int 
                 102 
                 CAGACTTTGCTACCAGGACTTGCT 
               
               
                   
               
            
           
           
               
               
               
            
               
                 MSH2 
                 Sequencing Primers internal instead of tagged primer in that direction 
                   
               
            
           
           
               
               
               
               
               
            
               
                 2 
                 MSH2-2seq-s2-int 
                 292 
                 GGAGCAAAGAATCTGCAGAGTGTT 
                   
               
               
                   
               
               
                 5 
                 MSH2-5seq-as2-int 
                 293 
                 CTGAAAAAGGTTAAGGGCTCTGACT 
               
               
                   
               
            
           
           
               
               
            
               
                 rev. 091703 AK 
                   
               
               
                 rev. 112003 AK 
               
               
                 rev. 021304 AK 
               
               
                 rev. 030604 AK 
               
               
                 rev. 032604 AK 
               
               
                   
               
               
                 note old name for exon 14-16 in brackets 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE F 
               
               
                   
               
               
                 EXTENSION PRODUCTS GENERATED FOR TTGE ASSAY 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Clamp region sense corresponds to: 
                   
               
               
                 5′ CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG (SEQ ID NO.: 344) 
               
               
                   
               
               
                 Clamp region rev. complement 
               
               
                 5′ CGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG (SEQ ID NO.: 345) 
               
               
                   
               
               
                 MSH2 2B-3 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGgGAGCAAAG 
               
               
                 AATCTGCAGAGTGTTGTGCTTAGTAAAATGAATTTTGAATCTTTTGTAAAAGA 
               
               
                 TCTTCTTCTGGTTCGTCAGTATAGAGTTGAAGTTTATAAGAATAGAGCTGGA 
               
               
                 AATAAGGCATCCAAGGAGAATgattggtatttggcatataaggtaatta 
               
               
                 (SEQ ID NO.: 346) 
               
               
                   
               
               
                 MSH2 2C 
               
               
                 ATAAGGCATCCAAGGAGAATGATTGGTATTTGGCATATAAGgtaattatcttccttttta 
               
               
                 atttacttatttttttaagagtagaaaaataaaaatgtgaagaatttaattgtgttttagtattttaagtagatCGGGC 
               
               
                 GGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG (SEQ ID NO.: 347) 
               
               
                   
               
               
                 MSH2 3A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGaacattttattaata 
               
               
                 aggttcatagagtttggatttttcctttttgcttataaaattttaaagtatgttcaagagtttgttaaatttttaaaattttattttt 
               
               
                 acttagGCTTCTCCTGGCAAT (SEQ ID NO.: 348) 
               
               
                   
               
               
                 MSH2 3B2 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGatttttacttagGC 
               
               
                 TTCTCCTGGCAATCTCTCTCAGTTTGAAGACATTCTCTTTGGTAACAATGATA 
               
               
                 TGTCAGCTTCCATTGGTGTTGTGGGTGTTAAAATGTCCGCAGTTGATGGCCA 
               
               
                 GAGACAGGTTGGAGTTGGGTATGTGGATTCCATACAGAGGAAACTAGGACT 
               
               
                 GTGTGAATTCCCTGATAATGATCAGTTCTCCAATCTTGAGGCTCTCCTCATC 
               
               
                 CAGATTGGACCAAAGGAATGTGTTTTACCCGGAGGAGAGACTGCTGGAGAC 
               
               
                 ATGGGGAAACTG (SEQ ID NO.: 349) 
               
               
                   
               
               
                 MSH2 3C 
               
               
                 AATGTGTTTTACCCGGAGGAGAGACTGCTGGAGACATGGGGAAACTGAGAC 
               
               
                 AGgtaagcaaattgagtctagtgatagaggagattccaggcctaggaaaggctctttaattgacatgatactgttt 
               
               
                 catttaagCGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 350) 
               
               
                   
               
               
                 MSH2 4A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGtccttttctcatagta 
               
               
                 gtttaaactatttctttcaaaatagATAATTCAAAGAGGAGGAATTCTGATCACAGAAAGA 
               
               
                 AAAAAAGCTGACTTTTCCACAAAAGACATTTATCAGGACCTCAA 
               
               
                 (SEQ ID NO.: 351) 
               
               
                   
               
               
                 MSH2 4A2 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGtttctttcaaaatag 
               
               
                 ATAATTCAAAGAGGAGGAATTCTGATCACAGAAAGAAAAAAAGCTGACTTTT 
               
               
                 CCACAAAAGACATTTATCAGGACCTCAACCGGTTGTTGAAAGGCAAAA 
               
               
                 (SEQ ID NO.: 352) 
               
               
                   
               
               
                 MSH2 4B2 
               
               
                 ATTTATCAGGACCTCAACCGGTTGTTGAAAGGCAAAAAGGGAGAGCAGATG 
               
               
                 AATAGTGCTGTATTGCCAGAAATGGAGAATCAGgtacatggattataaatgtgaattacaC 
               
               
                 GGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 353) 
               
               
                   
               
               
                 MSH2 4C 
               
               
                 ATTGCCAGAAATGGAGAATCAGgtacatggattataaatgtgaattacaatatatataatgtaaata 
               
               
                 tgtCGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 354) 
               
               
                   
               
               
                 MSH2 5A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGtcattttgcatttgttt 
               
               
                 tttaaaatctttagaactggatccagtggtatagaaatcttcgatttttaaattcttaattttagGTTGCAGTTTC 
               
               
                 ATCACTGTCTGCGGTAATCAAG 
               
               
                 (SEQ ID NO.: 355) 
               
               
                   
               
               
                 MSH2 5B 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGcttcgatttttaaatt 
               
               
                 cttaattttagGTTGCAGTTTCATCACTGTCTGCGGTAATCAAGTTTTTAGAACTCT 
               
               
                 TATCAGATGATTCCAACTTTGGACAGTTTGAACTGACTACTTTTGACTTCAGC 
               
               
                 CAGTATATGAAATTGGATATTGCAGCAGTCAGAGCCCTTAACCTTTTTCAGgt 
               
               
                 (SEQ ID NO.: 356) 
               
               
                   
               
               
                 MSH2 6A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGgtttttcatggcgta 
               
               
                 gtaaggttttcactaatgagcttgccattctttctattttattttttgtttactagGGTTCTGTTGAAGATACCA 
               
               
                 CTGGCTCTCAGT (SEQ ID NO.: 357) 
               
               
                   
               
               
                 MSH2 6B2 
               
               
                 tttactagGGTTCTGTTGAAGATACCACTGGCTCTCAGTCTCTGGCTGCCTTGCT 
               
               
                 GAATAAGTGTAAAACCCCTCAAGGACAAAGACTTGTTAACCAGTGGATTAAG 
               
               
                 CAGCCTCTCATGGATAAGAACAGAATAGAGGAGAGgtatCGGGCGGGGGCG 
               
               
                 GCGGGGCGGGCGCGGGGCGCGGCGGGCG (SEQ ID NO.: 358) 
               
               
                   
               
               
                 MSH2 6C 
               
               
                 TCAAGGACAAAGACTTGTTAACCAGTGGATTAAGCAGCCTCTCATGGATAAG 
               
               
                 AACAGAATAGAGGAGAGgtatgttattagtttatactttcgttagttttatgtaacctgcagttacccacatg 
               
               
                 attataccacttattCGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 359) 
               
               
                   
               
               
                 MSH2 7A2 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGgttgagacttacgt 
               
               
                 gcttagttgataaattttaattttatactaaaatattttacattaattcaagttaatttatttcagATTGAATTTAGT 
               
               
                 GGAAGCTTTTGTAGAAGATGCAGAATTG (SEQ ID NO.: 360) 
               
               
                   
               
               
                 MSH2 7B2 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGatttatttcagATT 
               
               
                 GAATTTAGTGGAAGCTTTTGTAGAAGATGCAGAATTGAGGCAGACTTTACAA 
               
               
                 GAAGATTTACTTCGTCGATTCCCAGATCTTAACCGACTTGCCAAGAAGTTTC 
               
               
                 AAAGACAAGCAGCAAACT (SEQ ID NO.: 361) 
               
               
                   
               
               
                 MSH2 7C3 
               
               
                 GACTTGCCAAGAAGTTTCAAAGACAAGCAGCAAACTTACAAGATTGTTACCG 
               
               
                 ACTCTATCAGGGTATAAATCAACTACCTAATGTTATACAGGCTCTGGAAAAA 
               
               
                 CATGAAGgtaacaagtgattttgtttttttgttttccttcaactcatacaatatatacttggcaatgtgctgtcctcata 
               
               
                 aagttggtggtggtgactcaCGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGG 
               
               
                 CGGGCG (SEQ ID NO.: 362) 
               
               
                   
               
               
                 MSH2 8A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGtttggatcaaatga 
               
               
                 tgcttgtttatctcagtcaaaattttatgatttgtattctgtaaaatgagatctttttatttgtttgttttactactttcttttagGA 
               
               
                 AAACACCAGAAATTATTGTTGGCAGTTTTTGTGACTCCTCTTACTGAT 
               
               
                 (SEQ ID NO.: 363) 
               
               
                   
               
               
                 MSH2 8B 
               
               
                 TTGTGACTCCTCTTACTGATCTTCGTTCTGACTTCTCCAAGTTTCAGGAAATG 
               
               
                 ATAGAAACAACTTTAGATATGGATCAGgtatgcaatatactttttaatttaagcagtagttaCGG 
               
               
                 GCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 364) 
               
               
                   
               
               
                 MSH2 8C 
               
               
                 CTGACTTCTCCAAGTTTCAGGAAATGATAGAAACAACTTTAGATATGGATCA 
               
               
                 Ggtatgcaatatactttttaatttaagcagtagttatttttaaaaagcaaaggccactttaagaaagtttgtagatttttc 
               
               
                 tttttagtatctaattgtagcacCGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGG 
               
               
                 CGGGCG (SEQ ID NO.: 365) 
               
               
                   
               
               
                 MSH2 8D 
               
               
                 AGAAATTATTGTTGGCAGTTTTTGTGACTCCTCTTACTGATCTTCGTTCTGAC 
               
               
                 TTCTCCAAGTTTCAGGAAATGATAGAAACAACTTTAGATATGGATCAGgtatgca 
               
               
                 atCGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 366) 
               
               
                   
               
               
                 MSH2 9A2 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGaatatttgctttata 
               
               
                 atttctgtctttacccattatttataggattttgtcactttgttctgtttgcagGTGGAAAACCATGAATTCCT 
               
               
                 TGTAAAACCTTCATTTGATCCTAATCTCAGTGAATTAAGAGAAATAATGAATG 
               
               
                 ACTTGGAAAAGAAGATGCAGTCAACATTAATAAGTGCAGCCAGAGATCTTGg 
               
               
                 taagaatgggtcattggaggttggaataattct (SEQ ID NO.: 367) 
               
               
                   
               
               
                 MSH2 10A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGgaattacattgaa 
               
               
                 aaatggtagtaggtatttatggaatactttttcttttcttcttgattatcaagGCTTGGACCCTGGCAAACA 
               
               
                 GATTAA (SEQ ID NO.: 368) 
               
               
                   
               
               
                 MSH2 10B2 
               
               
                 tcttcttgattatcaagGCTTGGACCCTGGCAAACAGATTAAACTGGATTCCAGTGCA 
               
               
                 CAGTTTGGATATTACTTTCGTGTAACCTGTAAGGAAGAAAAAGTCCTTCGTA 
               
               
                 ACAATAAAAACTTTAGTACTGTAGATATCCAGAAGAATGGTGTTACGGGCGG 
               
               
                 GGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 369) 
               
               
                   
               
               
                 MSH2 10C3 
               
               
                 TGCACAGTTTGGATATTACTTTCGTGTAACCTGTAAGGAAGAAAAAGTCCTT 
               
               
                 CGTAACAATAAAAACTTTAGTACTGTAGATATCCAGAAGAATGGTGTTAAATT 
               
               
                 TACCAACAGgtttgcaagtcgttattatatttttaaccctttattaattccctaaatgctctaacatgatgtgaatgtt 
               
               
                 ctatgataagttttacCGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGG 
               
               
                 CG (SEQ ID NO.: 370) 
               
               
                   
               
               
                 MSH2 11A2 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGtttggatatgtttca 
               
               
                 cgtagtacacattgcttctagtacacattttaatatttttaataaaactgttatttcgatttgcagCAAATTGACTT 
               
               
                 CTTTAAATGAAGAGTATACCAAAAATAAAACAGAATATGAAGAAGCCCAGGA 
               
               
                 TGCCATTGTTAAAG (SEQ ID NO.: 371) 
               
               
                   
               
               
                 MSH2 11B2 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGgCAAATTGA 
               
               
                 CTTCTTTAAATGAAGAGTATACCAAAAATAAAACAGAATATGAAGAAGCCCA 
               
               
                 GGATGCCATTGTTAAAGAAATTGTCAATATTTCTTCAGgtaaacttaatagaactaata 
               
               
                 atgttctgaatgtcacctggcttttggtaacagaagaaaaatcatgatatttgaagtgtgttttgttattttcgcaagcc 
               
               
                 at (SEQ ID NO.: 372) 
               
               
                   
               
               
                 MSH2 12A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGaggaaatgggtttt 
               
               
                 gaattcccaaatggggggattaaatgtatttttacggcttatatctgtttattattcagtattcctgtgtacattttctgttttt 
               
               
                 atttttatacagGCTATGTAGAACCAATGCAGACACTCAATGATGTGTTAGCTC 
               
               
                 (SEQ ID NO.: 373) 
               
               
                   
               
               
                 MSH2 12B2 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGatttttatacagGC 
               
               
                 TATGTAGAACCAATGCAGACACTCAATGATGTGTTAGCTCAGCTAGATGCTG 
               
               
                 TTGTCAGCTTTGCTCACGTGTCAAATGGAGCACCTGTTCCATATGT 
               
               
                 (SEQ ID NO.: 374) 
               
               
                   
               
               
                 MSH2 12C 
               
               
                 TGGAGCACCTGTTCCATATGTACGACCAGCCATTTTGGAGAAAAGGACAAGG 
               
               
                 AAGAATTATATTAAAAGCATCCAGGCATGCTTGTGTTGAAGTTCAAGATGAA 
               
               
                 ATTGCATTTATTCCTAATGACGTATACTTTGAAAAAGATAAACAGATGTTCCA 
               
               
                 CATCATTACTGgtaaaaaacctggtttttgggctttgtgggggtaacgttttgttCGGGCGGGGGCG 
               
               
                 GCGGGGCGGGCGCGGGGCGCGGCGGGCG (SEQ ID NO.: 375) 
               
               
                   
               
               
                 MSH2 12E 
               
               
                 cagctttgctcacgtgtcaaaTGGAGCACCTGTTCCATATGTACGACCAGCCATTTTGG 
               
               
                 AGAAAGGACAAGGAAGAATTATATTAAAAGCATCCAGGCATGCTTGTGTTGA 
               
               
                 AGTTCAAGATGCGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGG 
               
               
                 GCG (SEQ ID NO.: 376) 
               
               
                   
               
               
                 MSH2 13A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGaggactaacaat 
               
               
                 ccatttattagtagcagaaagaagtttaaaatcttgctttctgatataatttgttttgtagGCCCCAATATGGG 
               
               
                 AGGTAAATCAACATATATTCGACAAACTGGGGTGATAGTACTCATGGCCCA 
               
               
                 (SEQ ID NO.: 377) 
               
               
                   
               
               
                 MSH2 13B 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGATGGGAGGT 
               
               
                 AAATCAACATATATTCGACAAACTGGGGTGATAGTACTCATGGCCCAAATTG 
               
               
                 GGTGTTTTGTGCCATGTGAGTCAGCAGAAGTGTCCATTGTGGACTGCATCTT 
               
               
                 AGCCCGAGTAGGGGCTGGTGACAGTCAATTGAAAGGAGTC 
               
               
                 (SEQ ID NO.: 378) 
               
               
                   
               
               
                 MSH2 13C5 
               
               
                 TTGTGGACTGCATCTTAGCCCGAGTAGGGGCTGGTGACAGTCAATTGAAAG 
               
               
                 GAGTCTCCACGTTCATGGCTGAAATGTTGGAAACTGCTTCTATCCTCAGgtaa 
               
               
                 gtgcatctcctagtcccttgaagatagaaatgtatgtctctgtcctgtgaCGGGCGGGGGCGGCGGG 
               
               
                 GCGGGCGCGGGGCGCGGCGGGCG (SEQ ID NO.: 379) 
               
               
                   
               
               
                 MSH2 14A3 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGgtatgtgtatgttac 
               
               
                 cacattttatgtgatgggaaatttcatgtaattatgtgcttcagGTCTGCAACCAAAGATTCATTAAT 
               
               
                 AATCATAGATGAATTGGGAAGAGGAACTTCTACCTACGATGGATTTGGGTTA 
               
               
                 GCATGGGCTATATCAGAATACATTGCAACAAAGATTGGTGCTTTTTGCATGT 
               
               
                 TTGCAACCCATTTTCATGAACTTACTGCCTTGGCCAATCAGATACCAACTGTT 
               
               
                 AATAATCTACATGTCACAGCACTCACCACTGAAGAGACCTTAACTA 
               
               
                 (SEQ ID NO.: 380) 
               
               
                   
               
               
                 MSH2 14B 
               
               
                 ATAATCTACATGTCACAGCACTCACCACTGAAGAGACCTTAACTATGCTTTAT 
               
               
                 CAGGTGAAGAAAGgtatgtactattggagtactctaaattcagaacttggtaatgggaaacttactaccctt 
               
               
                 gaaatcatcagtaattgccttattcCGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGC 
               
               
                 GGCGGGCG (SEQ ID NO.: 381) 
               
               
                   
               
               
                 MSH2 15A 
               
               
                 gtctcttctcatgctgtcccctcacgcttccccaaatttcttatagGTGTCTGTGATCAAAGTTTTGGG 
               
               
                 ATTCATGTTGCAGAGCTTGCTAATTTCCCTAAGCATGTAATAGAGTGTGCTA 
               
               
                 AACAGAAAGCCCTGGAACTTGAGGAGTTTCAGTATATTGGAGAATCGCAAG 
               
               
                 GATATGATATCATGGAACCAGCAGCAAAGAAGTGCTATCTGGAAAGAGAGgtt 
               
               
                 tgtcagtttgttttcatagtttaacttagcttctctattCGGGCGGGGGCGGCGGGGCGGGCGCG 
               
               
                 GGGCGCGGCGGGCG (SEQ ID NO.: 382) 
               
               
                   
               
               
                 MSH2 16A 
               
               
                 ttactaatgggacattcacatgtgtttcagCAAGGTGAAAAAATTATTCAGGAGTTCCTGTCC 
               
               
                 AAGGTGAAACAAATGCCCTTTACTGAAATGTCAGAAGAAAACATCACAATAA 
               
               
                 AGTTAAAACAGCTAAAAGCTGAAGTAATAGCAAAGAATAATAGCTTTGTAAAT 
               
               
                 GAAATCATTTCACGAATAAAAGTTACTACGTGAaaaatcccagtaatggaatgaaggtaa 
               
               
                 tattgataagctattgtCGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGG 
               
               
                 GCG (SEQ ID NO.: 383) 
               
               
                   
               
               
                 MLH1 
               
               
                 Clamp region sense corresponds to: 
               
               
                 5′ CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCG 
               
               
                 (SEQ ID NO.: 344) 
               
               
                   
               
               
                 Clamp region rev. complement 
               
               
                 5′ CGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 345) 
               
               
                   
               
               
                 MLH1 1A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGcaatagctgccgc 
               
               
                 tgaagggtggggctggatggcgtaagctacagctgaaggaagaacgtgagcacgaggcactgaggtgattg 
               
               
                 gctgaaggcacttccgttgagcatctagacgtttccttggctcttctggcgccaaaATGTCGTTCGTGGC 
               
               
                 AGGGGTTATTCGGCGGCTGGACGAGACAGTGGTGAACCGCATCGCGGCGG 
               
               
                 GGGAAGTTATCCAGCG (SEQ ID NO.: 384) 
               
               
                   
               
               
                 MLH1 1B 
               
               
                 GGCGGGGGAAGTTATCCAGCGGCCAGCTAATGCTATCAAAGAGATGATTGA 
               
               
                 GAACTGgtacggagggagtcgagccgggctcacttaagggctacgacttaacgggccgcgtcactcaatg 
               
               
                 gcgcg CGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 385) 
               
               
                   
               
               
                 MLH1 1C 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCAAAGAGAT 
               
               
                 GATTGAGAACTGgtacggagggagtcgagccgggctcacttaagggctacgacttaacgggccgcgt 
               
               
                 cactcaatggcgcggacacgcctctttgcccgggcagaggcatg 
               
               
                 (SEQ ID NO.: 386) 
               
               
                   
               
               
                 MLH1 1D 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGggaagaacgtga 
               
               
                 gcacgaggcactgaggtgattggctgaaggcacttccgttgagcatctagacgtttccttggctcttctggcgcca 
               
               
                 aaATGTCGTTCGTGGCAGGGGTTATTCGGCGGCTGGACGAGACAGTGGTGA 
               
               
                 ACCGCATCGCGGCGGGGGAAGTTATCCAGCGgccagctaatg 
               
               
                 (SEQ ID NO.: 387) 
               
               
                   
               
               
                 MLH1 2A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGttatcattgcttggc 
               
               
                 tcatattaaaatatgtacattagagtagttgcagactgataaattattttctgtttgatttgccagTTTAGATGCA 
               
               
                 AAATCCACAAGTATTCAAGTGATTGTTAAAGAGGGAGGCCTGAAGTTGATTC 
               
               
                 AGATCCAAGACAA (SEQ ID NO.: 388) 
               
               
                   
               
               
                 MLH1 2B 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGGCAAAATCC 
               
               
                 ACAAGTATTCAAGTGATTGTTAAAGAGGGAGGCCTGAAGTTGATTCAGATCC 
               
               
                 AAGACAATGGCACCGGGATCAGGgtaagtaaaacctcaaagtagcaggatgtttgtgcgcttca 
               
               
                 tggaagagtcagg (SEQ ID NO.: 389) 
               
               
                   
               
               
                 MLH1 3A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGgggaattcaaag 
               
               
                 agatttggaaaaatgagtaacatgattatttactcatctttttggtatctaacagAAAGAAGATCTGGATA 
               
               
                 TTGTATGTGAAAGGTTCACTACTAGTAAACTGCAGTCCTTTGAGGATTTAGC 
               
               
                 CAGTATTTCTACCTATGGCTTTCGAGGTGAGgtaagctaaagattcaagaa 
               
               
                 (SEQ ID NO.: 390) 
               
               
                   
               
               
                 MLH1 3B 
               
               
                 ATATTGTATGTGAAAGGTTCACTACTAGTAAACTGCAGTCCTTTGAGGATTTA 
               
               
                 GCCAGTATTTCTACCTATGGCTTTCGAGGTGAGgtaagctaaagattcaagaaatgtgta 
               
               
                 aaatatcctcctgtgatgacattgtctgtcatttgttagtatgtatttctcaacatagataaataaggtttggtacCGG 
               
               
                 GCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 391) 
               
               
                   
               
               
                 MLH1 4A4 
               
               
                 ggtgaggtgacagtgggtgacccagcagtgagtttttctttcagtctattttcttttcttccttagGCTTTGGCCA 
               
               
                 GCATAAGCCATGTGGCTCATGTTACTATTACAACGAAAACAGCTGATGGAAA 
               
               
                 GTGTGCATACAGgtatagtgctgacttcttttactcatatatattcaCGGGCGGGGGCGGCGG 
               
               
                 GGCGGGCGCGGGGCGCGGCGGGCG (SEQ ID NO.: 392) 
               
               
                   
               
               
                 MLH1 4B2 
               
               
                 TCATGTTACTATTACAACGAAAACAGCTGATGGAAAGTGTGCATACAGgtatagt 
               
               
                 gctgacttcttttactcatatatattcattctgaaatgtattttttgcctaggtctcagagtaatcctgtctcaacaccagtg 
               
               
                 ttatcCGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 393) 
               
               
                   
               
               
                 MLH1 5A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGgggattagtatcta 
               
               
                 tctctctactggatattaatttgttatattttctcattagAGCAAGTTACTCAGATGGAAAACTGAAAG 
               
               
                 (SEQ ID NO.: 394) 
               
               
                   
               
               
                 MLH1 5B2 
               
               
                 CTGAAAGCCCCTCCTAAACCATGTGCTGGCAATCAAGGGACCCAGATCACG 
               
               
                 gtaagaatggtacatgggagagtaaattgttgaagctCGGGCGGGGGCGGCGGGGCGGGC 
               
               
                 GCGGGGCGCGGCGGGCG (SEQ ID NO.: 395) 
               
               
                   
               
               
                 MLH1 5C2 
               
               
                 GGGACCCAGATCACGgtaagaatggtacatgggagagtaaattgttgaagctttgtttgtataaatattg 
               
               
                 gaat CGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 396) 
               
               
                   
               
               
                 MLH1 5D 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGtttgttatattttctca 
               
               
                 ttagAGCAAGTTACTCAGATGGAAAACTGAAAGCCCCTCCTAAACCATGTGCT 
               
               
                 GGCAATCAAGGGACCCAGATCACGgtaagaat (SEQ ID NO.: 397) 
               
               
                   
               
               
                 MLH1 6-5 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGattcactatcttaa 
               
               
                 gacctcgcttttgccaggacatcttgggttttattttcaagtacttctatgaatttacaagaaaaatcaatcttctgttca 
               
               
                 gGTGGAGGACCTTTTTTACAACATAGCCACGAGGAGAAAAGCTTTAAAAAAT 
               
               
                 CCAAGTGAAGAATATGGGAAAATTTTGGAAGTTGTTGGCAGgtacagtccaaaatct 
               
               
                 gggagtgggtctctgagatttgtcatcaaagtaatgtgttctag 
               
               
                 (SEQ ID NO.: 398) 
               
               
                   
               
               
                 MLH1 7 
               
               
                 taactaaaagggggctctgacatctagtgtgtgtttttggcaactcttttcttactcttttgtttttcttttccagGTATTC 
               
               
                 AGTACACAATGCAGGCATTAGTTTCTCAGTTAAAAAAgtaagttcttggtttatgggggat 
               
               
                 ggttttgttttatgaaaagaaaaaaggggatttttaatagtttgctggtggagataaggttatgatgtttcagtctcagc 
               
               
                 catgagacaataaaCGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGG 
               
               
                 GCG (SEQ ID NO.: 399) 
               
               
                   
               
               
                 MLH1 8A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGgctggtggagata 
               
               
                 aggttatgatgtttcagtctcagccatgagacaataaatccttgtgtcttctgctgtttgtttatcagCAAGGAGA 
               
               
                 GACAGTAGCTGATGTTAGGACACTACCCAATGCCTCAACCGTGGACA 
               
               
                 (SEQ ID NO.: 400) 
               
               
                   
               
               
                 MLH1 8B2 (also has 4 bp miniclamp) 
               
               
                 GGGGGCAAGGAGAGACAGTAGCTGATGTTAGGACACTACCCAATGCCTCAA 
               
               
                 CCGTGGACAATATTCGCTCCATCTTTGGAAATGCTGTTAGTCGgtatgtcgataac 
               
               
                 ctatat CGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 401) 
               
               
                   
               
               
                 MLH1 8C2 
               
               
                 AAATGCTGTTAGTCGgtatgtcgataacctatataaaaaaatcttttacatttattatcttggtttatcattcca 
               
               
                 tcacattattttggaacctttcaagaCGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGC 
               
               
                 GGCGGGCG (SEQ ID NO.: 402) 
               
               
                   
               
               
                 MLH1 9A3 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGgtaatgtttgagtttt 
               
               
                 gagtattttcaaaagcttcagaatctcttttctaatagAGAACTGATAGAAATTGGATGTGAGGAT 
               
               
                 AAAACCCTAGCCTTCAAAATGAATGGTTACATATCCAATGCAAACTACTCAG 
               
               
                 TGAAGAAGTGCATCTTCTTACTCTTCATCAACCgtaagttaaaaagaaccacatgggaa 
               
               
                 atccactcacaggaaacacccacagggaattttatgggaccatggaaaaatttctg 
               
               
                 (SEQ ID NO.: 403) 
               
               
                   
               
               
                 MLH1 9B 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGcaaagttagtttat 
               
               
                 gggaaggaaccttgtgtttttaaattctgattcttttgtaatgtttgagttttgagtattttcaaaagcttcagaatctcttttc 
               
               
                 taatagAGAACTGATAGAAATTGGATGTGAGGATAAAACCCTAGCCTTCAAAAT 
               
               
                 GAATGGTTACATATCCAATGCAAACTACTCAGTGAAGAAGTGCATCTTCTTA 
               
               
                 CTCTTC (SEQ ID NO.: 404) 
               
               
                   
               
               
                 MLH1 9C 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCTTCAAAAT 
               
               
                 GAATGGTTACATATCCAATGCAAACTACTCAGTGAAGAAGTGCATCTTCTTA 
               
               
                 CTCTTCATCAACCgtaagttaaaaagaaccacatgggaaatccactcacaggaaacacccacaggg 
               
               
                 aat (SEQ ID NO.: 405) 
               
               
                   
               
               
                 MLH1 10 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGtgaatgtacacct 
               
               
                 gtgacctcacccctcaggacagttttgaactggttgctttctttttattgtttagATCGTCTGGTAGAATCAA 
               
               
                 CTTCCTTGAGAAAGCCATAGAAACAGTGTATGCAGCCTATTTGCCCAAAAA 
               
               
                 CACACACCCATTCCTGTACCTCAGgtaatgtagcaccaaactcctcaaccaagactcacaagg 
               
               
                 aacagatgttcta (SEQ ID NO.: 406) 
               
               
                   
               
               
                 MLH1 11A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGttgaccactgtgtc 
               
               
                 atctggcctcaaatcttctggccaccacatacaccatatgtgggctttttctccccctcccactatctaaggtaattgtt 
               
               
                 ctctcttattttcctgacagTTTAGAAATCAGTCCCCAGAATGTGGATGTTAATGTGCAC 
               
               
                 CCCACAAAGCATGAAGTTCACTTCCTGCAC (SEQ ID NO.: 407) 
               
               
                   
               
               
                 MLH1 11B 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCAGAATGTG 
               
               
                 GATGTTAATGTGCACCCCACAAAGCATGAAGTTCACTTCCTGCACGAGGAG 
               
               
                 AGCATCCTGGAGCGGGTGCAGCAGCACATCGAGAGCAAGCTCCTGGGCTC 
               
               
                 CAATTCCTCC (SEQ ID NO.: 408) 
               
               
                   
               
               
                 MLH1 11C4 
               
               
                 cagcagcacatcgagagcaagctcctgggctccaattcctccaggatgtacttcacccaggtcagggcgcttct 
               
               
                 catccagctacttctctggggcctttgaaatgtgcccggccagacgtgagagcccagatCGGGCGGGGG 
               
               
                 CGGCGGGGCGGGCGCGGGGCGCGGCGGGCG (SEQ ID NO.: 409) 
               
               
                   
               
               
                 MLH1 12B 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGttttttttaatacagA 
               
               
                 CTTTGCTACCAGGACTTGCTGGCCCCTCTGGGGAGATGGTTAAATCCACAA 
               
               
                 CAAGTCTGACCTCGTCTTCTACTTCTGGAAGTAGTGATAAGGTCTATGCCCA 
               
               
                 CCAGATGGTTCGTACAGATTCCCGGGAACAGAAGCTTGATGCATTTCTGCA 
               
               
                 GCCTCTGAGCAAACCCCTGTCCAGTCAGCCCCAGGCCATTGTCAC 
               
               
                 (SEQ ID NO.: 410) 
               
               
                   
               
               
                 MLH1 12C 
               
               
                 CATTTCTGCAGCCTCTGAGCAAACCCCTGTCCAGTCAGCCCCAGGCCATTG 
               
               
                 TCACAGAGGATAAGACAGATATTTCTAGTGGCAGGGCTAGGCAGCAAGATG 
               
               
                 AGGAGATGCTTGAACTCCCAGCCCCTGCTGAAGTGGCTGCCAAAAACGGG 
               
               
                 CGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 411) 
               
               
                   
               
               
                 MLH1 12D3 
               
               
                 AGCCCCTGCTGAAGTGGCTGCCAAAAATCAGAGCTTGGAGGGGGATACAA 
               
               
                 CAAAGGGGACTTCAGAAATGTCAGAGAAGAGAGGACCTACTTCCAGCAACC 
               
               
                 CCAGgtatggccttttgggaaaagtacagcctacctcctttattctgtaataaaactgccttctCGGGCGGG 
               
               
                 GGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG (SEQ ID NO.: 412) 
               
               
                   
               
               
                 MLH1 12E 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGTCCAGTC 
               
               
                 AGCCCCAGGCCATTGTCACAGAGGATAAGACAGATATTTCTAGTGGCAGGG 
               
               
                 CTAGGCAGCAAGATGAGGAGATGCTTGAACTCCCAGCCCCTGCTGAAGTG 
               
               
                 GCTGCCAAAAATCAGAG (SEQ ID NO.: 413) 
               
               
                   
               
               
                 MLH1 13A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGaatttggctaagttt 
               
               
                 aaaaacaagaataataatgatctgcacttccttttcttcattgcagAAAGAGACATCGGGAAGATTC 
               
               
                 TGATGTGGAAATGGTGGAAGATGATTCC (SEQ ID NO.: 414) 
               
               
                   
               
               
                 MLH1 13B3 (also has 12 bp miniclamp) 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCattgcagAAA 
               
               
                 GAGACATCGGGAAGATTCTGATGTGGAAATGGTGGAAGATGATTCCCGAAA 
               
               
                 GGAAATGACTGCAGCTTGTACCCCCCGGAGAAGGATCATTAACCTCACGCG 
               
               
                 GCGGGCG (SEQ ID NO.: 415) 
               
               
                   
               
               
                 MLH1 13C 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGATTCCCG 
               
               
                 AAAGGAAATGACTGCAGCTTGTACCCCCCGGAGAAGGATCATTAACCTCAC 
               
               
                 TAGTGTTTTGAGTCTCCAGGAAGAAATTAATGAGCAGGGACATGAGGgtacgta 
               
               
                 aacgctgtggcctg (SEQ ID NO.: 416) 
               
               
                   
               
               
                 MLH1 13D 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGATTAACCTC 
               
               
                 ACTAGTGTTTTGAGTCTCCAGGAAGAAATTAATGAGCAGGGACATGAGGgtac 
               
               
                 gtaaacgctgtggcctgcctgggatgcatagggcctca 
               
               
                 (SEQ ID NO.: 417) 
               
               
                   
               
               
                 MLH1 14A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGggtcaatgaagtg 
               
               
                 gggttggtaggattctattacttacctgttttttggttttattttttgttttgcagTTCTCCGGGAGATGTTGCA 
               
               
                 TAACCACTCCTTCGTGG (SEQ ID NO.: 418) 
               
               
                   
               
               
                 MLH1 14B 
               
               
                 agTTCTCCGGGAGATGTTGCATAACCACTCCTTCGTGGGCTGTGTGAATCCT 
               
               
                 CAGTGGGCCTTGGCACAGCATCAAACCAAGTTATACCTTCTCAACACCACC 
               
               
                 AAGCTTAGgtaaatcagctgagtgtgtgaacaagcagagctactacaacaatggtccagggagcacagg 
               
               
                 cacaaaagctaaggagagcagcatgaggtaCGGGCGGGGGCGGCGGGGCGGGCGCG 
               
               
                 GGGCGCGGCGGGCG (SEQ ID NO.: 419) 
               
               
                   
               
               
                 MLH1 15 
               
               
                 ttcagggattacttctcccattttgtcccaactggttgtatctcaagcatgaattcagcttttccttaaagtcacttcattttt 
               
               
                 attttcagTGAAGAACTGTTCTACCAGATACTCATTTATGATTTTGCCAATTTTGG 
               
               
                 TGTTCTCAGGTTATCGgtaagtttagatccttttcacttctgaaatttcaactgatcgtttctgaaaatagta 
               
               
                 gctctccactaatatcttatttgtagtatgttaaatttttcCGGGCGGGGGCGGCGGGGCGGGCGC 
               
               
                 GGGGCGCGGCGGGCG (SEQ ID NO.: 420) 
               
               
                   
               
               
                 MLH1 16A 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGgccattctgatagt 
               
               
                 ggattcttgggaattcaggcttcatttggatgctccgttaaagcttgctccttcatgttcttgcttcttcctagGAGCC 
               
               
                 AGCACCGCTCTTTGACC (SEQ ID NO.: 421) 
               
               
                   
               
               
                 MLH1 16B 
               
               
                 GCACCGCTCTTTGACCTTGCCATGCTTGCCTTAGATAGTCCAGAGAGTGGC 
               
               
                 TGGACAGAGGAAGATGGTCCCAAAGAAGGACTTGCTGAATACATTGTTGAG 
               
               
                 TTTCTGAAGAAGAAGGCTGAGATGCTTGCAGACTATTTCTCTTTGGAAATTG 
               
               
                 ATGAGgtgtgacagccattcttatacCGGGCGGGGGCGGCGGGGCGGGCGCGGGGC 
               
               
                 GCGGCGGGCG (SEQ ID NO.: 422) 
               
               
                   
               
               
                 MLH1 16C2 
               
               
                 GGCTGAGATGCTTGCAGACTATTTCTCTTTGGAAATTGATGAGgtgtgacagccat 
               
               
                 tcttatacttctgttgtattcttcaaataaaatttccagccgggtgcggtggctcatgCGGGCGGGGGCGG 
               
               
                 CGGGGCGGGCGCGGGGCGCGGCGGGCG (SEQ ID NO.: 423) 
               
               
                   
               
               
                 MLH1 17 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGtgtttaaactatga 
               
               
                 cagcattatttcttgttcccttgtcctttttcctgcaagcagGAAGGGAACCTGATTGGATTACCCCT 
               
               
                 TCTGATTGACAACTATGTGCCCCCTTTGGAGGGACTGCCTATCTTCATTCTT 
               
               
                 CGACTAGCCACTGAGgtcagtgatcaagcagatactaagcatttcggtacatgcatgtgtgctggagg 
               
               
                 gaaagggcaaatgaccacc (SEQ ID NO.: 424) 
               
               
                   
               
               
                 MLH1 18A2 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGtgtgatctccgttta 
               
               
                 gaatgagaatgtttaaattcgtacctattttgaggtattgaatttctttggaccagGTGAATTGGGACGAA 
               
               
                 GAAAAGGAATGTTTTGAAAGCCTCAGTAAAGAATGCGCTATGTTCTATTCCA 
               
               
                 TCCGGAAGCAGTACATATCTGAGGAGTCGACCCTCTCAG 
               
               
                 (SEQ ID NO.: 425) 
               
               
                   
               
               
                 MLH1 18B3 (also has 14 bp miniclamp) 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGTGCGCTATG 
               
               
                 TTCTATTCCATCCGGAAGCAGTACATATCTGAGGAGTCGACCCTCTCAGGC 
               
               
                 CAGCAGgtacagtggtgatgcacactggcaccccaggactagCGGGCGGGGGCGGC 
               
               
                 (SEQ ID NO.: 426) 
               
               
                   
               
               
                 MLH1 19A 
               
               
                 aagtctttccagacccagtgcacatcccatcagccaggacaccagtgtatgttgggatgcaaacagggaggctt 
               
               
                 atgacatctaatgtgttttccagagtgaAGTGCCTGGCTCCATTCCAAACTCCTGGAAGTG 
               
               
                 GACTGTGGAACACATTGTCTATAAAGCCTTGCGCTCACACATTCTGCCTCCT 
               
               
                 AAACATTTCACAGAAGATGGAAATATCCTGCAGCTTGCTAACCTGCCTGATC 
               
               
                 TATACACGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCGGGCG 
               
               
                 (SEQ ID NO.: 427) 
               
               
                   
               
               
                 MLH1 19B4 
               
               
                 AAGGCCTTGCGCTCACACATTCTGCCTCCTAAACATTTCACAGAAGATGGAA 
               
               
                 ATATCCTGCAGCTTGCTAACCTGCCTGATCTATACAAAGTCTTTGAGAGGTg 
               
               
                 GTTAAatatggttattCGGGCGGGGGCGGCGGGGCGGGCGCGGGGCGCGGCG 
               
               
                 GGCG (SEQ ID NO.: 428) 
               
               
                   
               
               
                 MLH1 19C (also has 7 bp miniclamp) 
               
               
                 CGCCCGCCGCGCCCCGCGCCCGCCCCGCCGCCCCCGCCCGCAGAAGATG 
               
               
                 GAAATATCCTGCAGCTTGCTAACCTGCCTGATCTATACAAAGTCTTTGAGAG 
               
               
                 GTGTTAAatatggttatttatgcactgtgggatgtgttcttctttctctgtattccgatacaaagtgttgtatcaaagt 
               
               
                 gtgatatacaCGGGCGG (SEQ ID NO.: 429) 
               
               
                   
               
               
                 All exons and clamps are in capital letters.