Abstract:
A method for detection, visualization and/or comparison of polynucleotide sequences of interest using specially designed sets of long and short probes that enhance resolution and simplify visualization and detection. Probe compositions useful for practicing this method and procedures for identifying useful probes and probe combinations. These methods are useful for the detection of genomic rearrangements, especially those associated with various diseases, disorders and conditions including cancer or for assessment of genomic rearrangements associated with therapy. The probe compositions may be used in kits for detection of genetic rearrangements or in companion diagnostic products or kits, such as kits for the diagnosis or assessment of predisposition to cancer such as colorectal cancer.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     The present application claims priority to U.S. Provisional Application No. 61/553,889, filed Oct. 31, 2011, the entire contents of which are incorporated herein by reference. On Oct. 30, 2012, an International Application (PCT/IB/12/02423; submission number 1000168921) was also filed with the same title, the entire contents of which are incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention relates to high-resolution, precise method for detecting genomic rearrangements in vitro using specially designed combinations of polynucleotide probes. The invention concerns accurate methods of detection and diagnosis of conditions, disorders and diseases associated with rearrangement of genomic DNA. 
     2. Description of the Related Art 
     The Multigenic Paradigm of Human Diseases 
     Advances in genetic analysis of human diseases have provided better insights into the molecular mechanisms contributing to disease initiation and progression. Previous associations were made between particular diseases and association and/or linkage disequilibrium to single base mutations in somatic genetic sequences or with particular single nucleotide polymorphisms (“SNPs”) in genomic DNA. Newer technologies have provided evidence that larger genetic alterations and rearrangements are associated with, or can constitute major causes of diseases, disorders or conditions having a genetic origin or basis. Disease associations have now moved from a monogenic to a multigenic paradigm where a disease&#39;s origins and progression is mainly linked to more than one single genetic mutation or origin. While these new insights provide better avenues for disease detection and treatments, they also highlight the need for combinatorial genetic analysis that goes beyond detection of single mutational events or SNPs by assessing disease associations with larger genomic rearrangements. Such combinatorial genetic analysis would provide a better, more precise and accurate diagnosis of a particular condition, disorder, disease or pathology, but would also help establishing a more appropriate medical survey, more accurate therapeutic decisions and interventions, as well as help in assessing the efficacy of such therapies and interventions. 
     Multigenic Causes of Genetic Disease 
     Genetic disorders manifesting the same or similar clinical signs and consequences can arise from both single and exclusive, or combined, mutations in various genes. Such mutations can fall within either the single base alteration and/or the class of large genetic rearrangements. A few examples of such genetic disorders are Fragile X syndrome (mutations and expansions in the FMR1 gene), Ataxia Telangectasia (single base pair mutations in either intronic and exonic sequences as well as deletions and translocations of the ATM gene), Seckel syndrome (mutations as well as large rearrangements in SCKL1, SCKL2, SCKL3, PCTN and ATR), autism (mutations as well as large rearrangements in GLO 1, MTF 1 and SLC11A3), Spinal Muscular Atrophy (mutations, deletions, transconversions as well as cis-duplications involving the SMN1 and SMN2 genes) and myotonic dystrophy (trinucleotide/tetranucleotide expansions in DM1 and DM2). 
     Multigenic Causes of Cancer Predisposition 
     In the case of cancer predisposition, there are several examples of familial cancer predisposition syndromes for which one can nominate several causative genes for which both single base alterations and/or large rearrangements were identified. 
     Breast and Ovary Cancer. Causative genes: BRCA1, BRCA2, ATM . . . 
     mutation type: higher proportion of point mutations identified so far. 
     Hereditary nonpolyposis colorectal cancer (Lynch syndroma). Causative genes: MSH2, MLH1, MSH6, EPCAM, . . . mutation type: equivalent proportion of point mutations has also been identified. 
     Multigenic Causes of Cancer Progression 
     Cancer progression is surely the human disease domain where the monogenic causative hypothesis was definitely ruled out since several years. First, the disease&#39;s initiation is strictly dependent of two molecular events (immortalizing and transforming) due to genetic alterations in at least two independent genes classified at either oncogene or tumor suppressor genes. Second, the disease&#39;s progression is linked to additional genetic alterations independent from the causative ones. Not only do these additional alterations play a role in cancer progression, they also were demonstrated to be the basis for appearance of resistance to therapy during treatments. Strikingly, in the list of cancer related genes, if extremely rare examples are only subject to discrete single base mutations (e.g., KRas or BRaf), the large majority is either subject to only large rearrangements (e.g., HER2, ALK . . . ) or to both single base mutations and large rearrangements (p53, c-myc, c-Met, EGFR . . . ). 
     The identification and characterization of multigenic conditions, disorders and diseases, including cancer, cardiovascular disease, diabetes and other heritable genetic conditions has been made difficult in part due to the imprecision of existing methods of molecular diagnosis. Molecular Combing is probably the sole approach allowing detecting all type of large genetic rearrangements (deletion, amplification, expansions, inversions, translocations . . . ) even in a complex and heterogeneous population (such as tumors). 
     High resolution barcodes allowing multiplex analysis of patients could help diagnostic at different level such as for patient stratification/classification and/or prognosis. 
     Multiplex High Resolution Barcodes for Identifying the Right Genetic Alterations as a Key Driver for Therapeutic Intervention 
     The Example of Myotonic Dystrophy 
     Myotonic Dystrophy (DM1) and Myotonic Dystrophy 2 (DM2) are two muscular dystrophies characterized by trinucleotide/tetranucleotide expansions in two different genes. If severe forms of DM1 can be clinically differentiated from DM2, milder DM1 forms are displayed extremely similar clinical signs than DM2. There is currently no cure for or treatment specific to myotonic dystrophy. However, DM1 patients exhibit Complications of the disease (heart problems, cataracts . . . ) not existing in DM2 that could can be treated but not cured. Differentiating DM1 and DM2 by the use of a multiplex assay of high resolution barcodes could thus help preventing and treating secondary effects 
     The Example of Hereditary Breast and Ovary Cancer 
     In certain countries (U.S.) detecting constitutional alterations in BRCA1/2 drives to therapeutic intervention (surgery/reconstitution). Thus, there is a clear need for an accurate diagnostic comprising all the potentially involved genes. Such a test could be made on the basis of a multiplex assay of high resolution barcodes comprising large chromosomal regions around genes known to be involved in this syndrome; BRCA1, BRCA2, ATM, ATR . . . 
     DNA Damage and Response Inhibitors Example 
     Synthetic lethality became a strong reality for therapeutic decision to include Cancer patients in specific protocols/regimens. One of the first examples was given with the demonstration that Breast cancer patients with BRCA deficiency exhibit a higher sensitivity to PARP inhibitors, a new category of drug acting on DNA Damage and Response pathway. More recently, this was extended to other type of inhibitors in this category such as ATM inhibitors but also to more traditional anti-cancer drugs including all types of DNA polymerase and replication inhibitors. 
     Not only does this concept extended to other inhibitors, but it was also demonstrated that it could be extended to other types of cancers such as lung and metastatic melanoma. 
     Here, a multiplex high resolution barcode will allow detection of genetic alteration in genes involved in DNA damage and response that could help predicting sensitivity to this class of inhibitors. A list of such genes could include BRCA1, BRCA2, ATM, ATR, MSH2, MLH1, MSH6, EPCAM . . . 
     The Lung Cancer Example 
     Numerous alterations involved in lung cancer could be multiplexed for a better patient classification such as: 
     LOH/Deletion (P53, STK11, LKB1, BRG1, KLF6); 
     Amplification (FGFR1, MET, EGFR, HER2 . . . ); 
     Translocation: (ALK); 
     All these genetic alteration are associated to therapeutic treatments: 
     P53: Nutlin (low doses Actinomycin D produce similar effects) 
     FGFR1: Masitinib, PD173074, SU5402 TK1258 AZD4547 . . . 
     MET: GSK1363089, ARQ197, SGX523, XL184 . . . 
     EGFR: Tarceva, Erbitux, Vectibix . . . 
     HER2: Herceptin, Lapatinib . . . 
     ALK: Crizotinib 
     As at least 30% of NSCLCs were demonstrated to be dependent on at least one of these mutations, defining the genetic profile of the tumor could help driving therapeutic options. This could be made possible by designing multiplex assays combining high resolution barcodes covering this major genetic loci. 
     Localization of (Genetic) Sequences of Interest 
     Genetic sequence is the most fundamental information to synthesize functional protein. Alteration of genetic sequence sometimes results in loss of functional protein synthesis. In addition to alteration of genetic sequence, loss or gain of genetic sequence (copy number variation, CNV) also can be problematic for homeostasis of cellular activity. For example, loss of (functional) anti-tumor protein (p53) or gain of proto-oncogene (c-myc) results in cancer-prone cell. When such mutation happens (or exists) in germ cell, this mutation spreads whole cell in an individual who is either carrier or patient of genetic disease, or has a predisposition to cancer. The germline mutation can be heritable. These days CNV becomes more and more important to understand in the field of genetics (ref 1). However, copy number count alone is not always sufficient and it is often critical to establish the actual location of sequence elements. This is strikingly the case for e.g. balanced translocations. DNA sequencing and CNV detection methods such as array-based comparative genomic hybridization (aCGH) and quantitative PCR generally cannot detect these balanced mutations because these methods assess whether the sequence and the copy number are correct or not. FISH and its extended forms such as fiber-FISH or molecular combing can address these balanced mutations with different resolutions and precisions depending on methods. 
     Resolution and Precision 
     The use of BAC/PAC/cosmid probes on targeted regions was successfully conducted to detect large (a few kb to tens of kb) genomic rearrangements (ref 2). In these approaches, the minimum size of detectable events (e.g., the size of the deleted or amplified sequence), hereafter designated as the “resolution” of such an assay, is limited due to the large standard deviation involved in measuring probes or gaps of tens of kilobases. Indeed, in such assays the standard deviation of measurements increases with the length of the measured element. For example, a 40 kb-probe is measured with a standard deviation of ˜5 kb. Thus, if 16 measurements of a given probe are made on a slide, the precision on the size of the probe obtained as the mean value of measurements is in the order of magnitude of 2.5 kb (Considering the distribution is gaussian, and the precision is the half-width of the confidence interval, i.e. 2.sd/√n where sd=standard deviation and n=number of measurements). For a 10 kb-probe, where the standard deviation is ˜2 kb, the precision would be ˜1 kb. This illustrates the fact that shorter probes allow for better (lower) resolution. 
     Besides, the location of such an event (the position of the extremities of the event) may be defined with a precision (hereafter the location precision) limited by the size of the probe or gap within which it occurs: e.g. if a 40 kb probe is estimated to measure 39 kb in a sample, one can conclude that a 1 kb deletion occurred somewhere within the probe, with no further precision—thus, somewhere in a 40 kb genomic region. If the same 1 kb deletion had occurred within a 10 kb probe, the location of that deletion would be known with a better precision, as the range would be reduced to a 10 kb genomic region. Therefore, the smaller the probes and gaps, the better the location precision. 
     There are limits to small probes: (i) below a certain size, they become difficult to detect; (ii) they involve more complex color schemes (as there are relatively more probes); (iii) there are more distinct probes to cover a given region, and the experiments are therefore more expensive and time-consuming; (iv) most importantly, fast and reliable identification of probes, whether by a human operator or a piece of software, is easier with longer probes, as they are more readily distinguished from background. Indeed, background is mainly constituted of roughly circular fluorescent spots. When large enough, the shape of these spots allows to one to easily distinguish them from probes. However, when their size is small enough, they appear difficult to distinguish from small probes. 
     In operating conditions according to the invention, probes shorter than ˜3 kb are detected with a diminished efficiency. Within the 3-10 kb range, the standard deviation of measurements varies little, and there is therefore little benefit in resolution with the shorter probes within this range. Therefore, this range is usually considered to be a good compromise for probe size. However, in cases where probes are close enough (less than 10 kb gaps), smaller probes (within the 500-3000 bp range) are still useful, as they will be detected in at least a fraction of signals and the presence of the corresponding sequences may therefore be established with certainty. It was also found that detection of isolated probes longer than 12 kb (preferably longer than 14 kb) is more reliable, whether for a human operator or for automatic detection software. 
     Exclusion of Repeats 
     Eukaryotic genomic DNA contains various repetitive sequences, i.e., sequences that appear more than once (and more than statistically predicted based on their length and base content) in a normal haploid genome. Among these, some appear with very high frequency (tens of thousands to millions of copies). In human genomic DNA, the most abundant of these is the Alu family, which has ˜1,000,000 copies constituting ˜10% of the genome. In any hybridization procedure involving human genomic DNA, it is expected that probes carrying such repeats would hybridize on numerous targets, generating non-specific signal from regions throughout the genome. Other types of repetitive sequences exist, with lower frequency, and often more specific localization. The number of copies and repeat sequence length may vary widely, as well as the degree of homology. Beta-satellite sequences, for example, are present in multiple copies (hundreds to thousands), usually as tandem repeat arrays comprising hundreds of copies of the same 50-100 bp long sequence, specifically localized in a limited number of loci. Strategies to get rid of the non-specific signals depend on the type of procedure and probe. Schematically, when probes are very short sequences of DNA (oligonucleotides, typically less than 100 bp), as in aCGH procedures, the sequence of the oligonucleotides is chosen to be free of repetitive sequences, by comparison with repetitive sequences found in databases. This strategy is only practical for very short probes, as short sequences free of repetitive sequences are relatively abundant, but unpractical for longer probes, as long stretches completely devoid of repetitive elements are rare (although this has been adapted to longer FISH probes, in an approach that suffers multiple drawbacks, see below). Besides, even for short probes, it constrains the design of probes heavily and some genomic regions, rich in repetitive sequences, have lower density of coverage (and thus lower resolution of events) due to this constraint. 
     When probes are longer (typically PCR products or cloned DNA inserts—1 to 150 kb), in Southern Blot or in FISH procedures, non-labeled competitive DNA, enriched in repetitive elements such as Alu repeats (usually Cot-1 DNA), is added in large excess along with the labeled probe. Competition of unlabelled probes on the repetitive sequences minimizes the hybridization of labeled probes. This strategy is expensive and since the competitor DNA is not purely made of repetitive sequences, competition also occurs on the unique sequences for which the probes were designed, thus limiting the amount of competitor DNA that may be used. Therefore, the efficiency of this approach is limited. 
     An alternative approach for longer probes has been proposed by Knoll and collaborators (U.S. Pat. No. 7,014,997), resembling the strategy usually adopted for oligonucleotides: probes are chosen within sequence intervals devoid from repetitive elements. This strategy is based on bioinformatics analysis of the regions of interest and exclusion of known repetitive sequences by comparison with sequence databases. However, this approach has several limitations: prior knowledge of the repetitive sequences is required, which can be a problem e.g. in species where such knowledge is unavailable. More importantly, intervals longer than 2 kb devoid of repetitive sequences appear only once in 20-30 kb on average and are unevenly distributed (Considering the distribution is gaussian, and the precision is the half-width of the confidence interval, i.e. 2.sd/√n where sd=standard deviation and n=number o) so the design of probes would be highly constrained, impairing the possibility to design a high-resolution code. This would prove especially difficult in repeat-rich regions, and/or regions where pseudogenes are located next to homologous genes of interest—such low-copy repetitive sequences being also excluded with the strategy from Knoll and co (ref 3). Since regions targeted in rearrangement tests, e.g., for diagnostics purposes, often display these features, this approach is not suitable for the design of high-resolution barcodes and especially not if such a code is to be used for diagnostics purposes. Distinctions between this approach and the invention are disclosed in more detail below. 
     BRIEF SUMMARY OF THE INVENTION 
     The present invention concerns the field of the in vitro diagnosis and detection of genetic rearrangements and is related to a method to identify or detect genetic rearrangements in a biological sample to be tested which are already known or which are new and provide markers for example of diseases as cancers or metabolic or foetal genetic diseases. The invention is characterized by using compositions containing purified or synthesized nucleic acid molecules (polynucleotides) having nucleotide sequences selected as short sequences with a length of less than 10 Kb and associated in the said method with other different nucleic acid molecules (polynucleotides) having nucleotide sequences non-overlapping with the former ones and having a size longer than 12 Kb. The selected nucleotide sequences (polynucleotides) used as probes are partly deleted of their natural frequently repeated sequences. The present invention concerns also improvements brought to the design of set of probe sequences for the detection of genetic rearrangements by hybridization as with fiber-FISH-like technologies such as Molecular Combing. The improvements described herein allow for high precision/high-resolution detection of rearrangements in time- and cost-efficient assays. This invention also relates to the use of probe sequences for diagnostics applications and companion diagnostics tests, to a method of detection of presence or absence of alterations in sequences and to a kit for the above uses. This is illustrated hereinafter with sets of nucleotide sequences corresponding to parts of at least two genes: MSH2 and MLH1 or to the regions of MSH2 and MLH1, whose mutations increase the risk of occurrence of human colorectal cancer. 
     The invention is related to the sets of polynucleotides or probes labeled or not which are specific of said genes. Presently, the detection of genetic rearrangements using current technologies is often insufficiently reliable for diagnostics use. Unlike most technologies used to detect genetic alterations, which suffer strong intrinsic limitations towards some types of rearrangements, direct technologies such as FISH or Fiber-FISH can intrinsically detect any type of rearrangements. Their use is mainly limited by their resolution. Molecular Combing, on the other hand, may reach sufficient resolution, but probe designs currently used fail to allow cost- and time-efficient high resolution analysis of rearrangements. 
     These improvements involve the combination within the same sets of probes of—typically shorter—probes designed to optimize the sensitive detection and precise measurement of rearrangements and—typically longer—probes to allow for fast and reliable detection of signals of interest when analyzing results. Alternative designs where the longer probes are replace with a combination of shorter probes having equivalent functions and effects are also disclosed. 
     Specific aspects of the invention based on the concept of combining small probes for resolution and long probes for ease of detection for the detection on one or more genomic region(s) of interest as disclosed in more detail below. 
     The invention thus concerns a method for detecting mutated or rearranged genomic polynucleotide (target) sequence comprising: 
     (a1) hybridizing a target genomic polynucleotide comprising one or more genomic region(s) of interest, where mutations or rearrangements are sought, to a set of short probes that bind to each region of interest without long gaps between the portions of the target sequence bound by the set of short probes, where on each genomic region a subset of short probes are selected so that when taken together they form a long contiguous stretch inside or outside the region of interest, and wherein the probes may optionally have frequent repetitive sequences removed and thus more generally are optionally devoid of such repetitive sequences; or 
     (a2) hybridizing a target genomic polynucleotide comprising one or more genomic region(s) of interest, where mutations or rearrangements are sought, to a set of short probes that bind to each region of interest without long gaps between the portions of the target sequence bound by the set of short probes and to one or more long (docking) probe(s) that bind to sequences near but outside of the region(s) of interest; wherein the sequence(s) of the long probe(s) does not overlap that of the short probes and wherein the short and/or long probes may optionally have frequent repetitive sequences removed and thus more generally are optionally devoid of such repetitive sequences; 
     (b) detecting the locations of hybridized probes on the genomic region(s) of interest; optionally, 
     (c) comparing the location of the hybridized probes on the target genomic polynucleotide sequence with one or more motifs based on the hybridization of said probes to a reference, control, normal, not mutated, or not rearranged genomic polynucleotide sequence; and optionally, 
     (d) correlating the presence of a mutated or rearranged genomic polynucleotide with a specific phenotype, disease, disorder, or condition. 
     The mutated or arranged genomic polynucleotide sequence can be obtained from a subject who has cancer or who is suspected to having cancer, for example, from a subject who has colorectal cancer or who is suspected of having colorectal cancer. In such a case, the short and long probes identify mutations or genomic rearrangements associated with colorectal cancer and a control or reference sample would not contain these mutations or rearrangements. The presence or risk of developing colorectal cancer is assessed by comparing a target genomic polynucleotide sequence with the reference and determining whether a mutation or rearrangement associated with colorectal cancer is present. This method can be practiced with specific probes corresponding to or derived from Probe sets 1, 2, 3 and 4. For colorectal cancer, a genomic region of interest can be selected from genes associated with this disease, such as MSH2, MLH1, MSH6, PMS2 or EPCAM. 
     Similarly, the method may be applied to samples obtained from subjects having or at risk of developing other kinds of cancer, such as breast cancer, ovary cancer, or lung cancer. The method may also be applied to samples obtained from subjects having or at risk of other kinds of diseases, disorders, or conditions, including cardiovascular disease, diabetes, neuromuscular disorders; such as myotonic dystrophy or spinal muscular atrophy or samples obtained from a subject who has, is suspected of having, or is suspected of being a carrier for a genetic or hereditary disease, disorder or condition, including known or unknown foetal genetic alterations. The sample can be obtained from a subject having a multigenic genetic or hereditary disease, disorder or condition or for a genetic or hereditary disease, disorder or condition associated with rearrangement of genomic DNA. 
     In some aspects of the invention, the sample will be obtained from a subject undergoing treatment for a disease, disorder or condition associated with a genomic or somatic genetic rearrangement and the results obtained are compared to results obtained at other time points before, during or after the termination of treatment. A companion test for evaluating the efficiency of a therapeutic drug on the mutated or rearranged nucleotide sequences of the gene or the region of the gene of interest can be performed using the short and long probes according to the invention. 
     Preferably, in the method described above, the hybridizing with the short and long probes in step a) will be performed simultaneously. 
     Preferably, the short probes range in length from 0.5 kb to 10 kb and the maximum size of the gaps between the short probes when they are bound to the target is 15 kb, preferably 12 kb and more preferably 10 kb. 
     The number of short probes employed in the method described above can range from 1, 2, 3 to 10, 15 or more. 
     The maximum size for the long probes is 150 kb and these probes preferably range from 12 kb to 40 kb in length. Preferably, in order to have “long probe(s) that bind to sequences near but outside of the region of interest”, distance between the long probes and the region of interest is no longer than 150 kb, and more preferably no longer than 75 kb and even more preferably no longer than 25 kb from the region of interest. The minimum size for a genomic region to be tested or targeted is 50 kb. The minimum number of regions of interest is one for a singleplex test and two or more for a multiplex test. Examples of combinations of short and/or long probes include at least one short (less than 10 kb) sequence and at least one non-overlapping long sequence (more than 15 kb), or at least one group of at least two short sequences, less than 10 kb each, which total group length is longer than 14 kb and less than 150 kb, hybridizing contiguously on the mutated or rearranged polynucleotide sequence. The short probes can comprise a set of contiguous probes that span a stretch of the genomic polynucleotide sequences inside or outside the region of interest that is at least 15 kb. 
     The long probes may have repetitive DNA sequences excluded. These repetitive sequences to be excluded would ordinarily appear more than once and more often than statistically predicted based on their length and base content, for example, repetitive sequences between 50 and 400 bp can be excluded, though shorter or longer repetitive sequences that decrease sensitivity or specificity of the method can be identified and excluded. An example of such a sequence is the repetitive Alu family DNA sequences. 
     According to an embodiment of the invention, in order for the probes, either short probes or long probes, to have repetitive sequences excluded, these probes are designed to hybridize in regions of the genome which are free of such repetitive sequences, i.e. which have less than 10% preferably less than 2% of the selected type(s) of repetitive sequences to be excluded. 
     In the method described above, the short and long probes are preferably fluorescently tagged and different components of the probe sets may be tagged with different labels, such as labels with different colors. Tagging provides one means to identify motifs or submotifs characteristic of a mutated or rearranged sequence. 
     Compositions or kits comprising a set of short probes or a combination of short and long probes as described herein and optionally one or more components for binding said probes to a polynucleotide, for performing molecular combing, and/or for detecting whether hybridization has occurred are also contemplated. For example, a composition containing the short and long probe(s) described above, wherein at least two of said probe sequences detect a genetic rearrangement by using Molecular Combing, said composition comprising either at least one short (&lt;12 kb) sequence and at least one non-overlapping long sequence (&gt;14 kb), or at least one group of at least two short sequences, less than 10 kb each, which total length is longer than 14 kb and less than 150 kb, hybridizing contiguously on the genetic target. The short probe(s) in such a composition may preferably range from 0.5 kb to 12 kb and the long probe(s) range from 14 kb to 40 kb. Frequent repetitive sequences described above may be removed from the probes. Examples of probe sequences are those that hybridize specifically on the MSH2 gene or in the region of the MSH2 gene or on the MLH1 gene or in the region of the MLH1 gene. Specific kinds of short probe sequence(s) where repetitive sequences have been removed include those selected from the group consisting of or comprising the sequences obtained by PCR amplification on human genomic DNA using the primer pairs described in Table 1 in the lines: 
     MSH2-v1 
     P3 (primer pairs P3a_MSH2-v1 to P3c_MSH2-v1, SEQ ID NO:21-26) 
     P4 (primer pairs P4a_MSH2-v1 to P4b_MSH2-v1, SEQ ID NO:27-30) 
     P5 (primer pairs P5a_MSH2-v1 to P5c_MSH2-v1, SEQ ID NO:31-36) P6 (primer pairs P6a_MSH2-v1 to P6b_MSH2-v1, SEQ ID NO:37-40) 
     P7 (primer pairs P7a_MSH2-v1 to P7c_MSH2-v1, SEQ ID NO:41-46) 
     P8 (primer pairs P8a_MSH2-v1 to P8b_MSH2-v1, SEQ ID NO:47-50) 
     P9 (primer pairs P9a_MSH2-v1 to P9c_MSH2-v1, SEQ ID NO:51-56) 
     P10 (primer pairs P10a_MSH2-v1 to P10b_MSH2-v1, SEQ ID NO:57-60) 
     MLH1-v1 
     P3 (primer pairs P3a_MLH1-v1 to P3d_MLH1-v1, SEQ ID NO:95-102) 
     P4 (primer pairs P4a_MLH1-v1 to P4b_MLH1-v1, SEQ ID NO:103-106) 
     P5 (primer pairs P5a_MLH1-v1 to P5b_MLH1-v1, SEQ ID NO:107-110) 
     P6 (primer pair P6a_MLH1-v1, SEQ ID NO:111-112) 
     P7 (primer pair P7a_MLH1-v1, SEQ ID NO:113-114 
     P8 (primer pairs P8a_MLH1-v1 to P8d_MLH1-v1, SEQ ID NO:115-122) 
     and the short probes may be used in combination with the long probe sequence(s) selected from the group consisting of or comprising the sequences obtained by PCR amplification on human genomic DNA using the primer pairs described in Table 1 in the lines 
     MSH2-v1 
     P11 (primer pairs P11a_MSH2-v1 to P11c_MSH2-v1, SEQ ID NO:61-66) 
     P12 (primer pairs P12a_MSH2-v1 to P12e_MSH2-v1, SEQ ID NO:67-76) 
     MLH1-v1 
     P9 (primer pairs P9a_MLH1-v1 to P9c_MLH1-v1, SEQ ID NO:123-128) 
     P10 (primer pairs P10a_MLH1-v1 to P10e_MLH1-v1, SEQ ID NO:129-138). 
     Specific kinds of contiguous short probe sequence(s) forming long stretches include those selected from the group consisting of or comprising the sequences obtained by PCR amplification on human genomic DNA using the primer pairs described in Table 1 in the lines: 
     MSH2-v2 
     PE1-2 (primer pairs PE1_MSH2-v2 to PE2_MSH2-v2, SEQ ID NO:163-166) and 
     PE3-6 (primer pairs PE3_MSH2-v2 to PE5-6_MSH2-v2, SEQ ID NO:167-172), together forming one stretch; 
     PE9 (primer pairs E9_MSH2-v2 and I9-10_MSH2-v2, SEQ ID NO:185-188), 
     PE10 (primer pair E10_MSH2-v2, SEQ ID NO:189-190), 
     PE11 (primer pairs E11_MSH2-v2 and I11-12_MSH2-v2, SEQ ID NO:191-194), 
     PE12-14 (primer pairs E12_MSH2-v2 and E13-14 MSH2-v2, SEQ ID NO:195-198) and 
     PE15-16 (primer pairs E15_MSH2-v2 and E16_MSH2-v2, SEQ ID NO:199-202), together forming one stretch; 
     MLH1-v2 
     PE1-2 (primer pairs E1_MLH1-v2 and E2_MLH1-v2, SEQ ID NO:227-230), 
     PE3-4 (primer pairs I23_MLH1-v2, E3_MLH1-v2 and E4_MLH1-v2, SEQ ID NO:231-236), 
     PE5-6 (primer pairs E5_MLH1-v2 and E6_MLH1-v2, SEQ ID NO:237-240), 
     PE7-9 (primer pairs E7-8_MLH1-v2 and E9_MLH1-v2, SEQ ID NO:241-244) and 
     PE10-11 (primer pairs E10_MLH1-v2 and E11_MLH1-v2, SEQ ID NO:245-248), together forming one stretch; 
     The primers designed for the purpose of preparing short probes of the invention may have a sequence of 20 to 40 nucleotides and comprise in their 3′ end a sequence of at least 20 contiguous nucleotides that base pairs with the target. The primer sequence thus may also comprise additional nucleotides that do not base pair with the target in its 5′ end. The nucleotides which do not base pair may be useful for the construction of the primers or for the cloning of the amplified sequence resulting from polymerization starting from the primers. In a particular embodiment the sequence of the primer that hybridizes to the target is longer than 20 nucleotides.
 
Molecular Combing is a powerful FISH-based technique for direct visualization of single DNA molecules that are attached, uniformly and irreversibly, to specially treated glass surfaces (Herrick and Bensimon, 2009); (Schurra and Bensimon, 2009). This technology considerably improves the structural and functional analysis of DNA across the genome and is capable of visualizing the entire genome at high resolution (in the kb range) in a single analysis.
 
Another embodiment of the invention is a method for designing a set of short probes or set of short and long probes as described above comprising:
 
     identifying a polynucleotide containing a genomic region of interest, 
     selecting long probe sequences outside of the genomic region of interest but within 100 kb of the closest probe in the region of interest, and preferably within 30 kb of the closest probe in the region of interest and optionally removing frequently repeated sequences from said long probe sequences, 
     selecting a short probe sequences from within the genomic region of interest so that no gaps longer than 20 kb, and preferably no gaps longer than 12 kb appear between the short probes; or selecting a series of short probes that together form a long continuous stretch that covers the genomic region of interest; 
     hybridizing the probes to a genomic polynucleotide comprising the genomic region of interest, 
     detecting the hybridized probes, and 
     determining which sets of probes form motifs that specifically identify the genomic sequence of interest from a reference genomic sequence. 
     The comparison of the location of the hybridized probes on the target genomic polynucleotide sequence with one or more motifs based on the hybridization of said probes to a reference, control, normal, not mutated, or not rearranged genomic polynucleotide sequence, as disclosed in the databanks or experimentally obtained on samples. 
     The techniques disclosed herein may be applied to diagnosis of disease as well as for the identification of genetic rearrangements associated with a disease, disorder or condition. They may also be used as companion diagnostics to study the responses of a subject or group of subjects who have particular rearrangements to therapy, responses to environmental agents, or the effects of lifestyle choices. Specifically, the diagnostic products and methods of the invention are useful for diagnosis and assessments for subjects having or at risk of developing colorectal cancer. High resolution barcodes allow multiplex analysis of patients for extended or expanded diagnosis at the levels of patient stratification/classification and prognosis. Thus, the techniques disclosed herein can also be used to predict the course and probably outcome of a disease, disorder or condition as well as the likelihood of progression, stability, or recovery. Multiplex high resolution barcodes also permit the identification of key genetic alterations in a subject that would benefit from a particular kind of therapy as well as a way to assess the reaction of a subject to a particular kind of therapy or therapeutic intervention. 
     Specific embodiments of the invention include the following, which embodiments are especially carried out in vitro. 
     A method for detecting mutated or rearranged genomic polynucleotide sequence comprising: (a1) hybridizing a target genomic polynucleotide comprising one or more genomic region(s) of interest, where mutations or rearrangements are sought, to a set of short probes that bind to each region of interest without long gaps between the portions of the target sequence bound by the set of short probes said set of short probes optionally including or being in combination with a (sub)set of short probes selected so that on each genomic region some of the short probes when taken together form a long contiguous stretch inside or outside the region of interest and where the short probes may optionally have frequent repetitive sequences removed; or (a2) hybridizing a target genomic polynucleotide comprising one or more genomic region(s) of interest, where mutations or rearrangements are sought, to a set of short probes that bind to each region of interest without long gaps between the portions of the target sequence bound by the set of short probes and to one or more long (docking) probe(s) that bind to sequences near but outside of the region(s) of interest; wherein the sequence(s) of the long probe(s) does not overlap that of the short probes and wherein the short and/or long probes may optionally have some or all of the frequently repeating sequences removed; (b) detecting the locations of hybridized probes on the genomic region(s) of interest; optionally, (c) comparing the location of the hybridized probes on the target genomic polynucleotide sequence with one or more motifs based on the hybridization of said probes to a reference, control, normal, not mutated, or not rearranged genomic polynucleotide)sequence; and optionally, and/or (d) correlating the presence of a mutated or rearranged genomic polynucleotide with a specific phenotype, disease, disorder, or condition. 
     The invention relates in particular to the method herein described wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has cancer or who is suspected of having cancer or who is susceptible to have a genetic predisposition to cancer. 
     The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has colorectal cancer or who is suspected of having colorectal cancer or who is susceptible to have a genetic predisposition to colorectal cancer, wherein said short and long probes identify mutations or genomic rearrangements associated with colorectal cancer, wherein said control, not mutated or normal genomic sequence is obtained from a subject not at risk for colorectal cancer and wherein the detection of a genomic rearrangement; and assessing presence of or risk of developing colorectal cancer when said genomic rearrangement is detected. In this method the probes can hybridize specifically on the MSH2 gene, in the region of the MSH2 gene, on the MLH1 gene, or in the region of the MLH1 gene. 
     The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has breast cancer or who is suspected to having breast cancer or who is susceptible to have a genetic predisposition to breast cancer. 
     The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has ovarian cancer or who is suspected to having ovarian cancer or who is susceptible to have a genetic predisposition to ovarian cancer. 
     The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has lung cancer or who is suspected to having lung cancer or who is susceptible to have a genetic predisposition to lung cancer. 
     The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has a cardiovascular disease, disorder or condition or who is suspected of having cardiovascular disease, disorder or condition or who is susceptible to have a genetic predisposition to cardiovascular disease, disorder or condition. 
     The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has a diabetes or who is suspected of having diabetes or who is susceptible to have a genetic predisposition to diabetes. 
     The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has a neuromuscular disorder or who is suspected of having a neuromuscular disorder. 
     The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has, is suspected of having, or is susceptible of being a carrier for a genetic or hereditary disease, disorder or condition. 
     The invention also relates in a particular embodiment to a method wherein the short and long probe sequences are specific to human genes or to human genomic regions associated with cancer, colorectal cancer or a foetal genetic alteration known or unknown when said region or gene is mutated or genetically rearranged. 
     The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject who has, is suspected of having, or is suspected of being a carrier for a multigenic genetic or hereditary disease, disorder or condition or for a genetic or hereditary disease, disorder or condition associated with rearrangement of genomic DNA. 
     The invention also relates in a particular embodiment to a method wherein the mutated or rearranged genomic polynucleotide sequence is obtained from a subject undergoing treatment for a disease, disorder or condition associated with a genomic inherited or acquired rearrangement and the results obtained are compared to results obtained at other time points before, during or after the termination of treatment. 
     The invention relates to method of any of the embodiments described herein, characterized by the following features taken individually or in any combination: the hybridizing with the short and long probes in (a2) is performed simultaneously; the short probes are 10 kb or less; and/or the short probe(s) comprise at least one short (less than 10 kb) sequence and at least one non-overlapping long sequence (more than 12 kb), or at least one group of at least two short sequences, less than 5, 6, 7, 8, 9 or 10 kb each, total group length is longer than 12 kb and less than 150 kb, hybridizing contiguously on the mutated or rearranged polynucleotide sequence. In these methods the short probes may comprise a set of contiguous probes that span a stretch of the genomic polynucleotide sequences inside or outside the region of interest that is at least 14 kb; and/or the long probe(s) may comprise one or more docking probes of more than 14 kb and less than 40 kb. The long probe(s) may have a length of at least 14 kb and bind to a polynucleotide sequence outside the region of interest. 
     Both the long and short probes may be designed to exclude frequently occurring repetitive DNA sequences. These repetitive DNA sequences, which may be excluded from the long and short probes, will generally appear more than once and more often than statistically predicted based on their length and base content. For example, a repetitive DNA sequence between 50 and 400 contiguous nucleotides in length, which appear more than once and more often than statistically predicted based on their length and base content, can be excluded from the short and/or long probe(s). One example of a repetitive sequence that can be excluded from the short and long probes is or are members of the repetitive Alu family DNA sequences. 
     In some embodiments of the invention the probes in (b) of the first embodiment are fluorescently tagged so that they can be detected fluorometrically. In other embodiments in b) each probe is tagged with one of two or more fluorescent tags. 
     According to other embodiments of the methods above, motifs or easily identifiable subsets of the probes are detected and compared instead of every probe sequence. 
     The methods described above may employ at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more short probes. These short probes may each have a length of least 500, 600, 700, 800, 900 or more base pairs (bp). In some embodiments of the methods above, the probes will be selected so that the gaps between short probes in the genomic region of interest are no more than 12 kb each. In further embodiments the short probes will bind to a single contiguous genomic region of interest or the short probes can be selected to bind to more than one non-contiguous genomic region of interest. The long probes used in the method above may be selected so as to be no more than 20, 30 or 40 kb. The or each of the genomic region(s) of interest in the methods described above can be selected to be longer than 50 kb. 
     Another embodiment of the invention is a kit comprising a set of short probes or a set of short and a set of long probe(s); and optionally one or more components for binding said probes to a polynucleotide, for performing molecular combing, and/or for detecting whether hybridization has occurred; (i) wherein the short probes comprise a set of probes that taken together bind to a long continuous stretch of the genomic region of interest; or (ii) wherein the long probes bind to sequences outside the genomic region of interest, do not overlap the short probe sequences; and optionally, where the repetitive sequences have been removed from the long and/or short probes. A kit of the invention is suitable and/or is specific for use in a method of the invention as disclosed herein. In a particular embodiment its short and/or long probes are characterized by the features described herein in relation with the methods. Such a kit may be employed for or contain instructions for the detection of genomic rearrangements associated with colorectal cancer or genetic predisposition to colorectal cancer; for the detection of genomic rearrangements associated with breast cancer or genetic predisposition to breast cancer; for the detection of genomic rearrangements associated with ovarian cancer or genetic predisposition to ovarian cancer; for the detection of genomic rearrangements associated with lung cancer or genetic predisposition to lung cancer. 
     Another embodiment of the invention is a composition containing the short, or short and long probe(s) described by the first embodiment above, wherein at least two of said probe sequences detect a genetic rearrangement by using Molecular Combing, said composition comprising either (a) at least one short (less than 10 kb) sequence and at least one non-overlapping long sequence (more than 14 kb), or (b) at least one group of at least two short sequences, less than 10 kb each, which total length is longer than 14 kb and less than 150 kb, hybridizing contiguously on the genetic target. In this composition the short probe(s) can range from 0.5 kb to 9 kb and the long probe(s) can range from 14 kb to 40 kb. The size of the short probes may range from 0.5 to 9 kb and at least 90% of the frequent repetitive sequences can be been removed from the short probe sequences. This composition may contain probes sequences that hybridize specifically on the MSH2 gene or in the region of the MSH2 gene or on the MLH1 gene or in the region of the MLH1 gene. 
     In yet another embodiment the invention involves a method for designing short and long probes described herein in relation to methods comprising (a) identifying a polynucleotide containing a genomic region of interest, (b) selecting long probe sequences outside of the genomic region of interest but within 100 kb of the closest probe within the region of interest and optionally removing frequently repeated sequences from the long probe sequences, (c) selecting a set of short probe sequences from within the genomic region of interest so that no gaps longer than 15 kb appear between the short probes; or selecting a series of short probes that together form a long continuous stretch that covers the genomic region of interest; (d) hybridizing the probes to a genomic polynucleotide comprising the genomic region of interest, (e) detecting the hybridized probes, and (f) determining which sets of probes form motifs that distinguish the genomic sequence of interest from a reference genomic sequence. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . (A) Dot-plot of MSH2 gene sequence on RP11-1084A21 BAC clone. (B) probe code v1 (without repetitive element) on RP11-1084A21. (C) probe code-v2 on RP11-1084A21. Diagonal lines are perfectly matched region of DNA between two sequences. Dots are representatives of repetitive elements. Higher density of dots (or grey band) are higher density of repetitive element. 
         FIG. 2 . Dot plot analysis of MLH1 region. (A) Dot-plot of MLH1 gene sequence on RP11-426N19 BAC clone. (B) probe code v1 (without repetitive element) on RP11-426N19. (C) probe code-v2 on RP11-426N19. 
         FIG. 3 . Designed probe set for MSH2 by exclusion of repetitive element. A) theoretical probe set (labeled in red and green in microscopy experiments represented here in grey and black, respectively), and position of exon (small numbered dots). (B) actual hybridization image corresponding to MSH2-v1 probe set. Original microscopy images consist of three channel images where each channel is the signal from a given fluorophore—these are acquired separately in the microscopy procedure. These channels are represented here as different shades on a grayscale: green probes are shown in black and red probes in gray, while the background (absence of signal) is white. The aspect ratio was not preserved, signals have been “widened” (i.e. stretched perpendicularly to the direction of the DNA fiber) in order to improve the visibility of the probes. 
         FIG. 4 . Designed probe set for MLH1 by exclusion of repetitive element. A) theoretical probe set (red and green), and position of exon (purple dot). (B) actual hybridization image corresponding to MLH1-v1 probe set. The same color conventions are used for diagrams and microscopy images as in panels A and B of  FIG. 3 . 
         FIG. 5 . Designed probe set for MSH2 with docking probes (v2). (A) theoretical probe set). B) actual hybridization image corresponding to MSH2-v2 probe set. The color conventions in this and the other 3-color microscopy images (and corresponding diagrams) is as follows: blue probes are represented in black, green probes in dark gray, red probes in light gray and the background is white. 
         FIG. 6 . Designed probe set for with docking probes (v2). (A) theoretical probe set). (B) actual hybridization image corresponding to MLH1-v1 probe set. The same color conventions are used for diagrams and microscopy images as in  FIG. 5 . 
         FIG. 7 . Validation of genomic rearrangement in MSH2 in LoVo cell line with v2 probe set. Sketches of both theoretical probe set ( FIG. 7A , top) and validated rearrangement ( FIG. 7A , bottom) by molecular combing. The photo ( FIG. 7B ) is the recurrent abnormal signal set which corresponding to deletion from exon 3 to exon 8 of MSH2 (as in  FIG. 7A , bottom). The same color conventions are used for diagrams and microscopy images as in  FIG. 5 .  FIG. 7C  provides the number of observations for each GMC. 
         FIG. 8 . Validation of genomic rearrangement in MLH1 in SK-OV-3 cell line with v2 probe set. Sketches of both theoretical probe set ( FIG. 8A , top) and validated rearrangement ( FIG. 8A , bottom) by molecular combing. The photo ( FIG. 8B ) is the representative (but few cases) signal set corresponding to the upper stream of MLH1 probe set (left side of theoretical probe set). The difference of observation number between MSH2 probe signal (normal) and MLH1 (a part of left side) clearly demonstrates that deletion of exon 4 to 19 in MLH1 is homozygous, (consistent with reference  7  ( FIG. 8C )). Molecular combing test also revealed that the breakpoint of deletion is larger than previously reported (downstream probes from exon 19 are all deleted). The same color conventions are used for diagrams and microscopy images as in  FIG. 5   
     
    
    
     Table 1. describes primer sequences and coordinates on human genomic DNA used for hybridization fragment synthesis to design the probes of the invention. These primers or variant therefore obtained by adding nucleotides in the ends of the described sequences and having up to 40 nucleotides, are part of the invention. 
     Table 2. Analysis of sequence of probe sets and their covering region. These sequences and the sets of probes that are disclosed in particular, are part of the invention. 
     Sequence of each of probe sets or region was subjected to RepeatMasker test and some of representative values are shown in the table. Sum length: sum up of sequence of all probes in each set. For MLH1 and MSH2 regions, this is the total length of each region. Repeat length: sum of sequences recognized as sorts of repeat in human genome. This includes sequences other than SINE. Total repeat. % of repeat length in sum length. SINE: % of sequences categorized as SINE in sum length. ALUs: % of sequences categorized as Alu family sequences in sum length. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The above described strategies, for the reasons mentioned, are unsuitable to design a high-resolution code for diagnostics applications using technologies such as molecular combing. 
     In the present invention, the probes are defined as follows: a short probe is a nucleic acid sequence complementary to a genomic sequence, which probe can be detected with a given marker (such as a fluorochrome) once hybridized on the genomic sequence. One probe may be either made of (i) one single fragment covering the whole sequence, or of (ii) several exactly contiguous fragments, and/or (iii) slightly overlapping fragments (with an overlap less than 250 bp) and/or (iv) fragments separated by a very short gap (less than 1000 bp). With such short overlaps or gaps, using Molecular combing in our current setup, the fragments appears almost contiguous. The distance may be adjusted depending on the specific technique and experimental conditions. For example, with less resolutive conditions, longer gaps (less than 2 kb) or overlaps may be tolerated, provided fragments separated by such a gap still appear contiguous. Under more resolutive conditions, gaps should be shorter (less than 200 bp) in order for the fragments to appear contiguous. Short probes range in size from 500 bp to 10 kb. 
     A long probe is a nucleic acid sequence complementary to a genomic sequence, which probe can be detected with a given marker (such as a fluorochrome) once hybridized on the genomic sequence. One probe may be either made of (i) one single fragment covering the whole sequence, or of (ii) several exactly contiguous fragments, and/or (iii) slightly overlapping fragments (with an overlap less than 250 bp) and/or (iv) fragments separated by a gap (less than 3.5 kb), provided that more than 70% of the target sequence stretch is covered by probes (i.e. provided the gaps represent less than 30% of the target sequence). With such overlaps or gaps, using Molecular combing in our current setup, the fragments are efficiently detected. The distance may be adjusted depending on the specific technique and experimental conditions. For example, with less resolutive conditions, longer gaps (less than 5 kb each, representing in total less than 50% of the sequence) or overlaps may be tolerated, provided fragments separated by such gaps are still detected efficiently. Also, under such conditions, longer probes should be used (more than 20 kb) to allow for efficient detection. Under more resolutive conditions, gaps should be shorter (less than 2 kb) in order for the fragments to be efficiently detected, and probes may still be efficiently detected with shorter size (more than 10 kb). Long probes range in size from 12 kb to 150 kb. 
     In the present invention, the size of probes reflects the length of the genomic sequence where the probe hybridizes, independently of the number of strands in the DNA molecules. Therefore, a probe may be described as 1 kb (1 kilobase=1000 bases) or, indifferently, as 1000 bp (base pairs): in both cases, the probe hybridizes over 1000 bases of one of the strands of the target DNA molecule (and, if the probe is double stranded, also on the 1000 complementary bases of the other strand of the target molecule). 
     In the present invention, a “barcode” designates a specific motif formed by a set of probes labeled with different markers, where the motif characteristics are the lengths of the probes in the set, the lengths of the gaps separating successive probes and the colors in which the probes are detected (or, more generally, the markers with which the probes are labeled). 
     If a high coverage barcode is to be designed for high resolution, probe and space lengths need to be roughly in the 0.5 kb to 10 kb range (see above). This makes it unpractical to design probes that completely exclude rearrangements, and yet are spaced closely enough for the code to allow high location precision. On the other hand, some non-specific hybridization (i.e. hybridization of [parts of] a probe on genomic regions that are not the designed target of that probe) of a probe is acceptable when using a code strategy for the reading of signals. Indeed, in applications such as Southern blot where the hybridization of a single probe is assessed or aCGH where hybridization of every probe is considered separately, the non-specific hybridization of probes on even a very limited number of regions may lead to completely unusable results. To a lesser extent, this is also the case with multiple-probe applications such as FISH, since the resolution of FISH is insufficient to distinguish genomic regions as far apart as several tens of megabases: a single non-specific hybridization would lead to unusable results if it were located close enough to the targeted region. 
     In molecular combing and other similar applications using a code strategy, the quantity of non-specifically hybridized probes is not in issue per se. If a probe (or fragments of a probe) hybridizes even multiple times outside the region of interest, it is unlikely it will recreate a motif sufficiently similar to the code to be confusing. Also, non-specific hybridization over short sequences (&lt;&lt;1 kb), even within the region of interest, would most likely not be detected, unless they are sufficiently clustered to generate a long (&gt;1 kb) stretch of non-specific hybridization. For the above reasons, the inventors have developed an alternative approach for the design of probes when the main issue is the design of a (several) high resolution code(s) in a (several) given genomic region(s). The main step of this approach relies only on the knowledge of the sequence of the region(s) themselves. When designing such a code, the major issue is to avoid significant non-specific hybridization within the regions of interest(s). Non-specific hybridization becomes an issue only if several probes display non-specific hybridization on neighboring sequences outside the region of interest. In the latter case, there is a risk that the pattern of probes resembles the original code, or a rearranged version of it, and this would likely lead to false conclusions. Although the invention described herein does not allow excluding such occurrences, this is relatively easily done once the method described herein has been used to exclude other non-specific hybridizations (see below). 
     The basis for this approach is the detection and exclusion of sequences that are repetitive within the region(s) of interest. For this, only the corresponding sequence(s) (the target sequence(s)) have to be known. One easy way to detect such repeats is the search for local sequence alignments within the target sequence(s), which can be done with e.g. a dot-plot comparison of each target sequence with itself and the other target sequences. A dot-plot is a graph with the two (sets of) sequences that are being compared forming the two axis, while dots are printed at every point where the coordinates correspond to a local homology. For example, if nucleotide x from sequence A (horizontal axis) matches nucleotide y from sequence B (vertical axis), then a dot will appear at the point with (x; y) coordinates. Graphically, local alignments appear as diagonal lines. Some more elaborate tools inspired from dot-plots are available, that compare short sequences (“words”, typically a few nucleotides/tens of nucleotides long) rather than single nucleotides, and display dots in various shades of gray depending on the extent of homology, thus allowing a direct visual reading of relaxed homologies (non-specific hybridization may well appear with incomplete homology). The comparison may also be done directly on both strands for one of the sequences, so homologies appear for both sense and reverse complement orientations. An example of such a tool is “Dotter” (ref. 4). 
     With these tools, very frequent repetitive sequences, such as Alu sequences in the Human genome, appear quite clearly, as they have local homologies with numerous other sequences within the target regions. Therefore, stretches with a high frequency of these sequences appear as a gray band (horizontal or vertical depending on whether the stretch is located on the vertical or horizontal axis). The exact appearance of these stretches with dot-plot display tools will depend on settings, and possibly word size. Settings were selected such that sequence stretches longer than 200 bp with more than 80% homology appear clearly and can be located with a roughly 10 bp precision. 
     A sequence of 200 bp or more that contains more than 10 significant homologous sequences (less than 1, 2, 3, 4, 5, 10, 15 or 20% nucleotide mismatch or insertion/deletion) within the regions of interest is a frequent repetitive sequence, prone to generate significant non-specific hybridization. It is generally possible to design probes in such a way that they are void of these frequent repetitive sequences, thus increasing the specificity and the high resolution of the present technology compared to the published previous methods. 
     “Docking” Probes 
     Although, as shown above, shorter probes make for more precise localization of breakpoints and measurement of deleted or amplified sequences, they are, generally speaking, more difficult to detect with fiber-fish techniques and molecular combing, as they appear as shorter stretches of signal, i.e., they are both smaller and less easy to distinguish from noise (fluorescent spots either unrelated to probes or to hybridization of probes). This is particularly true when considering automatic (computer-based) detection of signals. 
     It is therefore desirable to include longer probes in the code (for example, more than 12 kb and less than 150 kb, preferably more than 14 kb and less than 40 kb, in particular for the detection of genetic rearrangements in the regions of MSH2 or MLH1 genes). These probes would appear as actual lines (rather than spots), readily distinguishable from noise and easily detectable due to their size. Once the signals of interest are detected, the detection of other probes located on the same DNA fiber is easier. 
     This is especially true using technologies such as Molecular Combing where the linearity of the fibers implies the other probes, if any, are located in the alignment of the first probe. Therefore, the invention provides that the inclusion of longer (&gt;12 kb, preferably &gt;14 kb) probes in the set of probes is a step towards easier detection of signals of interest. Not all probes in the set need to be that long: in a fast and “rough” detection step, the long probes are sought, which allows the localization of signals of interest. These probes are called “docking probes” as they allow to “land” on the regions of interest efficiently. In a second step, the shorter probes are sought in the neighborhood of the docking probes (and more specifically in the case of Molecular Combing or related technologies, in the alignment of these probes). Although when performed by a human operator these steps can hardly be formally executed consecutively, if an operator may limit his search to longer probes, he can browse through images more rapidly, which would only allow him to detect these probes and spend more time on images where a docking probe is seen in order to look for other shorter probes. As the longer docking probes would locally diminish the location precision and the resolution of the code, it is preferable for them not to be located in the region where rearrangements are sought. This is possible if the probes are located near, but not in, the region of interest, e.g. at either end of this region. 
     If it is desirable to only consider complete signals in the analysis of a given region (i.e. signals covering the entire contiguous region), these longer probes may also be used to assess the integrity of the region: if there is a probe located at each end and both probes are present, no breakage of the fiber has occurred during the DNA preparation or stretching step. In cases where several non contiguous regions are analyzed in a single test, obviously each region has to have its “docking” probes in order to be correctly detected. 
     Continuous Stretch of Short Probes 
     An alternative to the “docking probes” approach above is to design the set of probes in such a way that at least some groups of shorter probes form a continuous stretch of signal. This is possible if probe sequences are adjacent. In that case, several probes, although short enough (less than 10 kb) to provide for sufficient resolution, may well combine to form a long enough (more than 14 kb) signal for fast and reliable detection. Indeed, if the operator may combine color channels to view images, this stretch would still appear as a long line rather than a spot, allowing its distinction from background noise. This is possible by using either common optical setups such as tri-color filters in fluorescence microscopy, or by using common image viewing software. In the case of automatic detection, it is also possible to use combined color information and therefore to make use of the very characteristic aspect of a multicolor line relatively to background spot-like noise. 
     Measurements 
     The probe designs described above likely lead to a large number of probes to be measured in a test. The usual approach for probe measurement is to measure all of the probes constituting a signal, as well as the gaps separating them. In a test with a large number of probes, the amount of work required for analyzing results is increased. In order to balance this, the invention relates to a more efficient designed approach for signal measurement. This approach consists in the measurement of subgroups of probes constituting easily recognizable motifs. The subgroups are two or several consecutive probes and the gaps between them, and possibly gaps at either end, chosen in order for their total length to remain within reasonably precise measurement range (10-30 kb). 
     There is likely to be a systematic bias in the measurement of digitalized images of fluorescent segments. Indeed, at the extremity of such a fragment, the intensity of the signal decreases gradually when moving away from the center, to reach the level of the background. Depending on where the operator/the software sets the threshold for the determination of the actual end there may be a systematic over- or under-estimation of the lengths. This bias is compensated for if the measured motifs have a probe at one end and a gap at the other. Therefore, it is preferable to design motifs in this way. 
     If a motif is found to have an abnormal length (different from the expected theoretical length) in a given sample, it remains possible to measure the probes and gaps within this motif in order to further precise the location of the rearrangement. With this approach, it is possible to measure in a fast and efficient way all of the signals for initial screening, while keeping the location precision allowed by small probes. The somewhat lower precision on measurements due to the larger size of the subgroups compared to the probes is essentially compensated for by the higher number of signals that can be measured within the same operator time. 
     Application to HNPCC—Rationale 
     Colorectal cancer is the 4th most frequent form of cancer in human and around 5% of the cancer is considered as a hereditary form. The most frequent form of hereditary colorectal cancer is known as Lynch syndrome, or HNPCC (hereditary non-polyposis colorectal cancer). HNPCC increases a lifetime risk of cancer development in up to 80% (lifetime risk is around 7% in normal population US). HNPCC also increases other cancers (endometrial, ovarian, stomach). 
     Genetic aspect of HNPCC is known as a result of mutation in some of Mismatch Repair (MMR) genes such as MSH2, MLH1, MSH6, PMS2, etc. MSH2 and MLH1 mutation accounts for more than 80% of all mutation of MMR genes in HNPCC. Both point mutation and large rearrangements are reported in mutation of those genes, and especially high % of large mutation in MSH2 is observed because of high level of small repetitive element in its genetic sequence. Today the molecular diagnosis is done after studies of familial cancer history, tumor characterization by microsatellite instability test. 
     Normally mutation one alleles of one of MMR genes is sufficient for molecular diagnosis of HNPCC. All HNPCC individuals have both wild and mutated genes. Point mutation of targeted MMR genes can be detected by sequencing of genes and current sequencing test investigates only the sequence of exons. In case of large rearrangements such as deletion and amplification (loss and gain of genetic elements, respectively), sequencing does not detect them because altered sequences do not exist, and frequently primer binding regions for sequencing are deleted. As a result, sequence information comes from only wild allele and gives false negative. Indeed, MSH2 and MLH1 genes are higher percentage of repetitive elements of SINE in their genetic sequence. To address this large rearrangement, the test should detect presence of deletion or amplification in the MMR genes. One approach is cartography of MMR genes with designed probes of hybridization. Causal large rearrangement has a wide range from sub-kb to loss of total gene (up to 100 kb). A given cartography has to be sensitive to this wide dynamic range of mutation. To cope with it specific probe design was done for MSH2 and MLH1 loci. 
     The present invention is also related to the detection of known or unknown genomic rearrangements. It is also related to kits containing probes according to the invention, for the detection of known or unknown genomic rearrangements and the associated pathologies, or associated predispositions to pathologies such as cancers or cardiovascular diseases for example. 
     EXAMPLES 
     Application to HNPCC—Materials and Method 
     Probe Design v1 
     Each probe (probe means continuous hybridization signal, can consist of multiple cloned DNA fragments, e.g., probe 1 of MSH2-v2 covers a 15 kb stretch and consists of five cloned DNA fragments of 3 kb. Since gap or overlap of each junction of these five fragments are smaller than resolution (&lt;50 bp), they are considered and indeed look like continuous single probe of 15 kb) on a region of gene sequence itself has a length between 3-6 kb. In case of larger rearrangement than probe or gap size, obvious change of color pattern of designed probe will be observed. As well as large rearrangement in probe region, such rearrangement is also detectable in gap region, meaning any rearrangement larger than 1 kb at any position in the target genes are detectable. This is a uniqueness of cartography method with high resolution probe hybridization. Other techniques (MLPA, aCGH) can detect only such rearrangement involving probe sequence. For genes with high frequency of large rearrangement such as MSH2 and MLH1, presence of repetitive element in their genetic sequence limits a freedom of probe design for the other technology. Inclusion of repetitive element sequence in their probe design increases false detection a lot, their probe designing has to be free of repetitive element in principle. 
     Probe sequence was chosen by a dot plot analysis. BAC clone sequence of each gene (RP11-1084A21 (Ch2:47,574,044-47,785,729 for MSH2, RP11-426N19 (Ch3: 36,992,516-37,161,490) for MLH1 was self-plotted and all grey bands region were excluded from the target region of PCR primer design. PCR primer set was designed in the target regions by Primer3plus PCR primer design tool (ref 6). A list of the primers&#39; sequence is shown in table 1A and B. Exclusion of Alu repeat was verified by both dot-plot analysis and RepeatMasker (http://www._repeatmasker.org).  FIG. 1B  and  FIG. 2B  show a lot less grey band on dot-plot of probe fragment sequence on BAC clone than dot-plot of gene (containing Alu repeat) on BAC clone. This indicates that sequence of designed probes does not include recurrent repetitive sequence in this target regions. RepeatMasker analysis (with default setting of web server) also clearly shows a dramatic reduction of % of Alu sequence in designed probe sequence. (table 2). 
     Probe Design v2 
     To facilitate “recognition” of barcodes on hybridization images, an alternative design of probe set (called v2) was done as said in “Docking” probe section. Design process is same as v1 except no exclusion of repetitive elements based on dot-plot. For v2 probe design, each probe was designed to have more than 3 kb length, close to limit to be recognized as “line”, and all exon sequences are covered by a probe stretch (no exons fall in gaps). Docking probes were designed on both extremities of each gene with 15-20 kb length. For MSH2-v2 code, specific probes covering EPCAM gene (see rationale part) was also included between two docking probes. DNA sequence of designed code v2 was subjected to dot-plot analysis to make sure that there is no segmental repeats inside of designed region ( FIGS. 1C and 2C ). 
     Cloning of Probe Fragments and Labeling for Hybridization Probe 
     Each fragment of probes was amplified by PCR, then the fragment was ligated into plasmid vector (pNEB193, pCR2.1-TOPO, pCRXL-TOPO). The ligation product was transformed into  E. coli  competent cells and end-sequences of cloned fragment were verified. Purified plasmid DNA set of each gene was separated into two (v1) or three (v2) gropes according to colors corresponding to theoretical barcodes ( FIG. 3A  and  FIG. 4A  for v1,  FIG. 5  and  FIG. 6  for v2 probe sets). Each group of plasmid DNA was labeled by random priming method. Either whole plasmids containing probe fragments&#39; sequence or PCR amplified probe fragments were used as a template for random priming. There are three haptens to be used for three color detection, biotin (Biot), digoxigenin (Dig) and Alexa Fluor 488 (A488). Biot-labeling was done by BioPrime DNA labeling system (Invitrogen) with manufacture&#39;s instruction. For Dig and A488 labeling, dNTP mixture in the kit was replaced with home-blend dNTP mixtures (either 0.1 mM Digoxigenin-11-dUTP (Roche applied science) for Dig labeling or 0.1 mM ChromaTide® Alexa Fluor® 488-7-OBEA-dCTP (Invitrogen) for A488 labeling, 0.1 mM unmodified equivalent (dTTP or dCTP) and 0.2 mM each of other three deoxynucleotides in final labeling reaction solution.). 
     Sample DNA Preparation 
     3 cell human cell lines were used for validation for large rearrangement detection in either MSH2 or MLH1. Cell line GM17939 was used as non-mutated sample. Cell line LoVo was used for MSH2 rearrangement validation, which is homozygous for deletion of exon 3-exon8 in MSH2. Another cell line SK-OV-3 was used for rearrangement validation of MLH1, which was reported as homozygous deletion of exon 4-exon 19 in MLH1. For each cell line, cell culture was prepared according to cell bank&#39;s instruction. Cultured cells were harvested (for LoVo and SK-OV-3 when 50-70% confluency) or collected by centrifuge (for GM17939 when between 300,000-400,000 cells/ml of medium. Cell pellet was resuspended in 1×PBS/Trypsin mixture to have 1,000,000 cells in 45 μl the cell suspension was mixed with an equal volume of 1.2% (w/v) NuSieve GTG agarose solution in 1×PBS (melted and equilibrated at 50° C. in advance). The cell/agarose mixture as poured into a well of gel plug mold, followed by gelification at 4° C. for 30 min. the gelified agarose plug was immersed in a mixture of 2 mg/ml of Proteinase K, 1% (w/v) of sarcosyl in 0.5M EDTA (pH8.0, 2500 for each plug). The agarose plug was incubated at 50° C. overnight. 
     Next day the incubated plug was washed in 1×TE (10 mM Tris-HCl, 1 mM EDTA, pH8.0) 3 times for 1 hour each. The DNA plug can be stored in 0.5mEDTA at 4° C. The washed plug was stained in 1000 of 33 μM YOYO-1 (Invitrogen) in TE40.2 (40 mM Tris-HCl, 2 mM EDTA pH8.0) for 1 hour in the dark. The stained plug was heated at 68° C. in 1 ml of combing buffer (0.5M MES pH5.5) for 20 min, then cooled at 42° C. 10 min prior to add 1.5 unit of beta agarase I (NEB). Beta agarase treatment was carried overnight at 42° C. in the dark. 
     The following day the treated DNA solution was poured into a combing reservoir and a level of the solution in the reservoir was adjusted with additional combing buffer. 
     Molecular Combing 
     The DNA solution was set on a Molecular Combing Machine (MCS, Genomic Vision). Molecular combing was performed on a silanized coverslips (Combicoverslips, Genomic Vision). The combed coverslips was fixed at 68° C. for 4 hours, then used for hybridization (or stored at −20° C. until use). 
     Hybridization and Detection of Probe 
     For one hybridization, 5 μl of each of labeled probe solutions (of both MSH2 and MLH1) was combined together and with 10 μg of sonicated herring or salmon sperm DNA and 10 μg of human Cot1-DNA (only for V2 probe sets), then purified by standard ethanol precipitation. The precipitate was resuspended with 20 μlof hybridization buffer (50% formamide, 2×SSC, 1% SDS and BlockAid blocking solution (Invitrogen)). The resuspended probe solution was set on a clean glass slide and covered with a DNA combed coverslip. The slide was heated at 90° C. for 5 min for co-denaturation of both probe and combed DNA then incubated at 37° C. overnight with an humidity for hybridization between labeled probes and combed DNA. 
     The hybridized coverslips was washed in 50% Formamid/2×SSC solution 3 times for 5 min each, followed by another 3 times washing with 2×SSC for 5 min each. The washed coveslips was then developed with two or three layers of fluorescently labeled antibodies or streptavidin. For each layer, antibodies for all haptens were diluted 25 times in BlockAid blocking solution (200 in final volume) and incubated for 20 min at 37° C. For Biot, Streptavidin Alexa Fluor 594 (Invitrogen) was used for the 1 st  and the 3 rd  layer, biotin conjugated-goat anti-streptavidin antibody was used for the 2 nd  layer. Fr Dig, mouse anti-Digoxin AMCA conjugated (Jackson immunoresearch) was for the 1 st  layer, rat anti-mouse AMCA conjugated (Jackson immunoresearch) conjugated was for the 2 nd , the goat anti-rat Alexa Fluore 350 conjugated (Invitrogen) was used for the 3 rd  layer. For A488, rabbit anti-Alexa Fluor 488 (Invitrogen) was used for the 1 st  layer, goat anti-rabbit Alexa Fluor 488 conjugated was used for the 2 nd  layer (no third antibody for A488). After 20 min incubation of each layer of antibody, the coverslip was washed in 2×SSC/1% Tween 20 washing solution 3 times for 5 min each at room temperature. After the washing of 3 rd  layer, the coverslip was rinsed in 1×PBS, followed by successive bath of 70, 90 and 100% ethanol for 1 min each. The coverslip was dried at room temperature prior to microscopy. 
     Signal Acquisition and Measurement 
     Fluorescent signal of developed antibody on the coverslip was obtained by standard epi-fluorescent microscope system or automated fluorescent microscope system (Image Xpress Micro, Molecular Devices) with custom scanning configuration for molecular combing signal. Every set of linearly aligned fluorescent signals and gaps was measured by ImageJ. Each measured set of signals (with color information) was subjected to pattern matching to determine position (if the set is a part of one of probe set) and orientation by comparison with the theoretical probe sets. All unclassified sets (did not match with any positions and orientations of theoretical probe sets) were subjected to similarity check between them to find whether recurrent abnormal pattern appears or not. 
     Application to HNPCC—Results 
       FIGS. 3B and 4B  are representative images of signal from hybridized DNA. Some of probes look like “dot” rather than “line” as expected from their length. There are some “random” spots on images of hybridization, but these spots do not interfere recognition of designed code. Although signals of some small probes (arrowed in  FIG. 3B , for example) is not evident to measure “length” of probe signals for size evaluation, measurement of “distance” between probe signals is possible and equivalent to measurement of the length of probe and gaps in normal probe set hybridization 
       FIGS. 5B and 6B  are the representative image of hybridization signal of barcodes-v2. Fluorescent signals are more continuous than the signals of barcodes-v1, and easier to find docking probes and measure the length of each probe and gap. These barcodes-v2 were used to visualize large genomic rearrangements of characterized cancer cell lines, LoVo and SK-OV-3 (ref. 5). 
       FIG. 7  is a result of hybridization of barcodes v2 on combed DNA from LoVo cell line; LoVo cell line is homozygous for deletion in MSH2 (from exon 3 to 8). Hybridization slide had many normal (identical to theoretical code) signal of MLH1 gene but none of normal MSH2 signals. Instead, there was a recurrent signal of truncated form of the normal MSH2 signal ( FIG. 7B ). By deduction from the truncated signals, this truncation results from loss of probes and gaps corresponding to ex3 to 8 of MSH2 gene. 
       FIG. 8  is a result of barcodes-v2 on SK-OV-3 cell line DNA, homozygous for deletion in MLH1 (from ex4 to 19). Among many normal MSH2 signals, only a few signals of part of MLH1 (from probe 1 to probe 3) were observed. This means a lack of following sequence of MLH1, which is consistent with reference. Moreover, a lack of the right (downstream of MLH1) docking probe indicates that this deletion affects beyond exon 19 of MLH1. 
     The sequences selected to detect predisposition to colorectal cancer linked to rearrangements in the MSH2 genomic region or the MLH1 genomic region are preferably chosen among the following nucleotide sequences and their corresponding complementary sequences and are described as: 
     The short probes covering the MSH2 gene region and constituting contiguous stretches (PE1-2 and PE3-6 (SEQ ID NO:354-358); PE9 to PE15-16 (SEQ ID NO:365-373) in table 1 under the header MSH2-v2) and the other short probes covering MSH2 gene region (PE7 and PE8, SEQ ID NO:359-364 in table 1 under the header MSH2-v2); the long probes neighboring the MSH2 gene (tPP1, EPCAM5′, EPCAM3′ (SEQ ID NO:342-353) and cPP1 (SEQ ID NO:374-378) in table 1 under the header MSH2-v2); the short probes covering the MLH1 gene region and constituting a contiguous stretch (PE1-2 to PE10-11, SEQ ID NO:386-396, in table 1 under the header MLH1-v2) and the other short probes covering MLH1 gene region (PE12-13, PE14-15 and PE16-19, SEQ ID NO:397-401, in table 1 under the header MLH1-v2); the long probes neighboring the MLH1 gene (tPP1 (SEQ ID NO:379-385) and cPP1 (SEQ ID NO:402-408) in table 1 under the header MLH1-v2). For example, these probes may be obtained by amplification of the fragments using the primers listed in Table 1 under the headers MSH2-v2 (SEQ ID NO:139-212) and MLH1-v2 (SEQ ID NO:213-272). 
     INCORPORATION BY REFERENCE  
     Each document, patent, patent application or patent publication cited by or referred to in this disclosure is incorporated by reference in its entirety, especially with respect to the specific subject matter surrounding the citation of the reference in the text. However, no admission is made that any such reference constitutes background art and the right to challenge the accuracy and pertinence of the cited documents is reserved. 
     
       
         
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Name 
                   
                 SEQ ID 
                   
                 SEQ ID 
                   
                   
                   
               
               
                 of 
                 Name of 
                 NO 
                 For/ 
                 NO 
                   
                   
                   
               
               
                 probe 
                 fragment 
                 (fragment) 
                 Rev 
                 (primer) 
                 Sequence (5′-3′) 
                 start 
                 end 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 MSH2-v1 
               
             
          
           
               
                 P1 
                 P1a_MSH2-v1 
                 273 
                 forward 
                 1 
                 TTCTTCCCAAGAGAGCCAAG 
                 47595911 
                 47595930 
               
               
                   
                   
                   
                 reverse 
                 2 
                 CTGTTTTGGAACCCCAAGTC 
                 47597074 
                 47597093 
               
               
                   
                 P1b_MSH2-v1 
                 274 
                 forward 
                 3 
                 GGCTTCAATCTGGGACTACG 
                 47598716 
                 47598735 
               
               
                   
                   
                   
                 reverse 
                 4 
                 GCTGTCACCGCCTCTTTTAC 
                 47599478 
                 47599497 
               
               
                   
                 P1c_MSH2-v1 
                 275 
                 forward 
                 5 
                 GCCAGGCACTTAGGCAGTAG 
                 47600433 
                 47600452 
               
               
                   
                   
                   
                 reverse 
                 6 
                 TTGGTCCTGACATCCTTTCC 
                 47601671 
                 47601690 
               
               
                   
                 P1d_MSH2-v1 
                 276 
                 forward 
                 7 
                 TTAGTTGAACAGGGCATGACAC 
                 47602097 
                 47602118 
               
               
                   
                   
                   
                 reverse 
                 8 
                 GGTAAAGGGGCCTGATGTC 
                 47602743 
                 47602761 
               
               
                   
                 P1e_MSH2-v1 
                 277 
                 forward 
                 9 
                 GAGCCTTGATGTTCCCTCTTAAC 
                 47603695 
                 47603717 
               
               
                   
                   
                   
                 reverse 
                 10 
                 ACCCAGATCCGAAACTGTTG 
                 47604324 
                 47604343 
               
               
                   
                 P1f_MSH2-v1 
                 278 
                 forward 
                 11 
                 CCGGCCTTACCTTTCATTTC 
                 47605735 
                 47605754 
               
               
                   
                   
                   
                 reverse 
                 12 
                 CCAGGATCCAGATCCAGTTG 
                 47606965 
                 47606984 
               
               
                 P2 
                 P2a_MSH2-v1 
                 279 
                 forward 
                 13 
                 GAGTTCCATGGCAGATCACC 
                 47612521 
                 47612540 
               
               
                   
                   
                   
                 reverse 
                 14 
                 GCAGCTTTCAATCACAAATCAG 
                 47614067 
                 47614088 
               
               
                   
                 P2b_MSH2-v1 
                 280 
                 forward 
                 15 
                 GAAGGGTTGGTCTTGCTGTC 
                 47615115 
                 47615134 
               
               
                   
                   
                   
                 reverse 
                 16 
                 ACCCTTTGCACCTCTCTGTG 
                 47615632 
                 47615651 
               
               
                   
                 P2c_MSH2-v1 
                 281 
                 forward 
                 17 
                 CCCGGTGTTGAATCATTTG 
                 47616079 
                 47616097 
               
               
                   
                   
                   
                 reverse 
                 18 
                 TTCAGCCCTGAAGGTAGAGG 
                 47617513 
                 47617532 
               
               
                   
                 P2d_MSH2-v1 
                 282 
                 forward 
                 19 
                 CTGGCCACTTTTTGGAAGAG 
                 47618884 
                 47618903 
               
               
                   
                   
                   
                 reverse 
                 20 
                 TGGGACGCAGAGTGATACAG 
                 47619394 
                 47619413 
               
               
                 P3 
                 P3a_MSH2-v1 
                 283 
                 forward 
                 21 
                 TTACTGGCGATCCTCAGAGC 
                 47629651 
                 47629670 
               
               
                   
                   
                   
                 reverse 
                 22 
                 AACGCCTCTTCCGTTGTATG 
                 47631623 
                 47631642 
               
               
                   
                 P3b_MSH2-v1 
                 284 
                 forward 
                 23 
                 GAAAGGACAGACCAAGTGCAG 
                 47632605 
                 47632625 
               
               
                   
                   
                   
                 reverse 
                 24 
                 AGCCTGTGCAGGGAAACTC 
                 47633083 
                 47633101 
               
               
                   
                 P3c_MSH2-v1 
                 285 
                 forward 
                 25 
                 AGTGGGATGCAGCTGAAAAG 
                 47633591 
                 47633610 
               
               
                   
                   
                   
                 reverse 
                 26 
                 CAACAGCATGGGAAAGATCC 
                 47635238 
                 47635257 
               
               
                 P4 
                 P4a_MSH2-v1 
                 286 
                 forward 
                 27 
                 TTGAAAGTTGGTCTTAGGAAGAGG 
                 47643286 
                 47643309 
               
               
                   
                   
                   
                 reverse 
                 28 
                 CCCAACAAACCTGGCTTTAG 
                 47644179 
                 47644198 
               
               
                   
                 P4b_MSH2-v1 
                 287 
                 forward 
                 29 
                 AGACGCCCAAAATCAACAAC 
                 47645155 
                 47645174 
               
               
                   
                   
                   
                 reverse 
                 30 
                 CCGCTTGCTGCTAAAAATTG 
                 47646042 
                 47646061 
               
               
                 P5 
                 P5a_MSH2-v1 
                 288 
                 forward 
                 31 
                 TGATTGCCAAGGAAGATTCAC 
                 47657647 
                 47657667 
               
               
                   
                   
                   
                 reverse 
                 32 
                 TGGAAGTAAATGCAGGTGCTC 
                 47658763 
                 47658783 
               
               
                   
                 P5b_MSH2-v1 
                 289 
                 forward 
                 33 
                 TCATTCTTGGGTGTTTCTCG 
                 47659578 
                 47659597 
               
               
                   
                   
                   
                 reverse 
                 34 
                 ATGGCGGTTTTGTGGAATAG 
                 47660015 
                 47660034 
               
               
                   
                 P5c_MSH2-v1 
                 290 
                 forward 
                 35 
                 GAGGGAGAGGGAACCTTTTG 
                 47661699 
                 47661718 
               
               
                   
                   
                   
                 reverse 
                 36 
                 GGGGACTATACCGCATTCAC 
                 47662243 
                 47662262 
               
               
                 P6 
                 P6a_MSH2-v1 
                 291 
                 forward 
                 37 
                 TGTTGATTCATGGGCATTTG 
                 47669651 
                 47669670 
               
               
                   
                   
                   
                 reverse 
                 38 
                 GCTGGGGAATCATGTATGAAG 
                 47671879 
                 47671899 
               
               
                   
                 P6b_MSH2-v1 
                 292 
                 forward 
                 39 
                 CATCAAGCACAGTTCCATTG 
                 47672243 
                 47672262 
               
               
                   
                   
                   
                 reverse 
                 40 
                 TTCTCTTTCCGTTTCCAGTG 
                 47673113 
                 47673132 
               
               
                 P7 
                 P7a_MSH2-v1 
                 293 
                 forward 
                 41 
                 GGAGCTTGGGAATTCAACTG 
                 47678126 
                 47678145 
               
               
                   
                   
                   
                 reverse 
                 42 
                 AGAAACGGGCATGTCATAGG 
                 47679330 
                 47679349 
               
               
                   
                 P7b_MSH2-v1 
                 294 
                 forward 
                 43 
                 CAGCCTACGTGCCCATTTC 
                 47679649 
                 47679667 
               
               
                   
                   
                   
                 reverse 
                 44 
                 TCAAAAGATGGCCAAAATGC 
                 47681179 
                 47681198 
               
               
                   
                 P7c_MSH2-v1 
                 295 
                 forward 
                 45 
                 GTGTTGCACCCATTAACTCG 
                 47681915 
                 47681934 
               
               
                   
                   
                   
                 reverse 
                 46 
                 AGCCTGGTGAGAGGTGACTG 
                 47684723 
                 47684742 
               
               
                 P8 
                 P8a_MSH2-v1 
                 296 
                 forward 
                 47 
                 CACGATGCCAGTCCAATTC 
                 47689478 
                 47689496 
               
               
                   
                   
                   
                 reverse 
                 48 
                 AAGGTGGACTTTAATGCAAAGG 
                 47690835 
                 47690856 
               
               
                   
                 P8b_MSH2-v1 
                 297 
                 forward 
                 49 
                 GGAGTGAGAGCGACACCTTG 
                 47691634 
                 47691653 
               
               
                   
                   
                   
                 reverse 
                 50 
                 CGACAGCTGACTGCTCTATGG 
                 47694068 
                 47694088 
               
               
                 P9 
                 P9a_MSH2-v1 
                 298 
                 forward 
                 51 
                 CACAATGGGAAAGGATGTAGC 
                 47701939 
                 47701959 
               
               
                   
                   
                   
                 reverse 
                 52 
                 CAGAGAAAAACACCCATGACC 
                 47704112 
                 47704132 
               
               
                   
                 P9b_MSH2-v1 
                 299 
                 forward 
                 53 
                 CACCGTGATCCTCCTTATTTC 
                 47704395 
                 47704415 
               
               
                   
                   
                   
                 reverse 
                 54 
                 GAACAAACAACGGATGAAAGG 
                 47704945 
                 47704965 
               
               
                   
                 P9c_MSH2-v1 
                 300 
                 forward 
                 55 
                 GTGGCATATCCTTCCCAATG 
                 47705311 
                 47705330 
               
               
                   
                   
                   
                 reverse 
                 56 
                 CCCCCAGACTGTGAATTAAGG 
                 47705787 
                 47705807 
               
               
                 P10 
                 P10a_MSH2-v1 
                 301 
                 forward 
                 57 
                 GATGCAGATCAGGGAAATGC 
                 47711630 
                 47711649 
               
               
                   
                   
                   
                 reverse 
                 58 
                 ATCTTGCTGGATGGACAAGG 
                 47715272 
                 47715291 
               
               
                   
                 P10b_MSH2-v1 
                 302 
                 forward 
                 59 
                 CTTAATCCTGAAAGGCAGGTG 
                 47715788 
                 47715808 
               
               
                   
                   
                   
                 reverse 
                 60 
                 TGTTTCTCAGGCAACCACAG 
                 47717266 
                 47717285 
               
               
                 P11 
                 P11a_MSH2-v1 
                 303 
                 forward 
                 61 
                 GAAACCACAGAATCGCCTTC 
                 47731087 
                 47731106 
               
               
                   
                   
                   
                 reverse 
                 62 
                 ACCTGGACAGTCCCACAGAC 
                 47733482 
                 47733501 
               
               
                   
                 P11b_MSH2-v1 
                 304 
                 forward 
                 63 
                 CAGTGCTTTTGCATCCTTCC 
                 47734903 
                 47734922 
               
               
                   
                   
                   
                 reverse 
                 64 
                 ATTTAATCCCCTGGCCAATC 
                 47741649 
                 47741668 
               
               
                   
                 P11c_MSH2-v1 
                 305 
                 forward 
                 65 
                 CACCTGTGCCCATCACATAG 
                 47742239 
                 47742258 
               
               
                   
                   
                   
                 reverse 
                 66 
                 GAGTCCCCTCTTGGAGAACC 
                 47747829 
                 47747848 
               
               
                 P12 
                 P12a_MSH2-v1 
                 306 
                 forward 
                 67 
                 AAAGCCATTTCCAGTGTCG 
                 47753989 
                 47754007 
               
               
                   
                   
                   
                 reverse 
                 68 
                 ATTGTGCAGCCAGAATTGAG 
                 47758158 
                 47758177 
               
               
                   
                 P12b_MSH2-v1 
                 307 
                 forward 
                 69 
                 TTCACAGCAAAGTGGCTCAG 
                 47760593 
                 47760612 
               
               
                   
                   
                   
                 reverse 
                 70 
                 GCTATTATGGGCTGCAAAGC 
                 47764302 
                 47764321 
               
               
                   
                 P12c_MSH2-v1 
                 308 
                 forward 
                 71 
                 TTCACTCCCAACAAGCACTG 
                 47764863 
                 47764882 
               
               
                   
                   
                   
                 reverse 
                 72 
                 TGCCCAGTCCTTTTTCACT 
                 47765618 
                 47765636 
               
               
                   
                 P12d_MSH2-v1 
                 309 
                 forward 
                 73 
                 AATCCCTCCTGCACACTTTC 
                 47765925 
                 47765944 
               
               
                   
                   
                   
                 reverse 
                 74 
                 AATGGATGCTTCCACTGTCC 
                 47767687 
                 47767706 
               
               
                   
                 P12e_MSH2-v1 
                 310 
                 forward 
                 75 
                 CCATCTGTGCAATTCCTTCC 
                 47768105 
                 47768124 
               
               
                   
                   
                   
                 reverse 
                 76 
                 GTTCAAAGGCAGAAGCCATC 
                 47769886 
                 47769905 
               
             
          
           
               
                 MLH1-v1 
               
             
          
           
               
                 P1 
                 P1a_MLH1-v1 
                 311 
                 forward 
                 77 
                 GTCTGGATTCTTTCACAATGTAGC 
                 37005551 
                 37005576 
               
               
                   
                   
                   
                 reverse 
                 78 
                 TGCCAATCTTCTCCTCTGTTC 
                 37006562 
                 37006582 
               
               
                   
                 P1b_MLH1-v1 
                 312 
                 forward 
                 79 
                 AACCACCCAATGTGTTCACC 
                 37006815 
                 37006836 
               
               
                   
                   
                   
                 reverse 
                 80 
                 GTTCATTCCTGCGAGTAGGC 
                 37007422 
                 37007441 
               
               
                   
                 P1c_MLH1-v1 
                 313 
                 forward 
                 81 
                 GCCAAAGGTGGAAAATGTTG 
                 37008987 
                 37009008 
               
               
                   
                   
                   
                 reverse 
                 82 
                 GCCTTCTTCATGAAAGCACTG 
                 37009873 
                 37009893 
               
               
                   
                 P1d_MLH1-v1 
                 314 
                 forward 
                 83 
                 CCAGAAGGTGGAAGCTACAG 
                 37011079 
                 37011100 
               
               
                   
                   
                   
                 reverse 
                 84 
                 TGGGGTCAATGAAGCAAG 
                 37011830 
                 37011847 
               
               
                   
                 P1e_MLH1-v1 
                 315 
                 forward 
                 85 
                 ACATCGACCCAGAAAGTTCC 
                 37012314 
                 37012335 
               
               
                   
                   
                   
                 reverse 
                 86 
                 AATGTGCTTCGTACCACTGC 
                 37012867 
                 37012886 
               
               
                   
                 P1f_MLH1-v1 
                 316 
                 forward 
                 87 
                 AGCGTGCCATTGTACTCTCC 
                 37013822 
                 37013843 
               
               
                   
                   
                   
                 reverse 
                 88 
                 TTTCTGAGCCCATGATTTCC 
                 37015267 
                 37015286 
               
               
                 P2 
                 P2a_MLH1-v1 
                 317 
                 forward 
                 89 
                 GTGCCCAGCTAGTTCCATTC 
                 37023623 
                 37023644 
               
               
                   
                   
                   
                 reverse 
                 90 
                 TCAAGAGCGCTAATCCCATC 
                 37025002 
                 37025021 
               
               
                   
                 P2b_MLH1-v1 
                 318 
                 forward 
                 91 
                 TGCACATGCTCACTGAAAGAC 
                 37026505 
                 37026527 
               
               
                   
                   
                   
                 reverse 
                 92 
                 TTTTGCCTGCAACTGACC 
                 37027818 
                 37027836 
               
               
                   
                 P2c_MLH1-v1 
                 319 
                 forward 
                 93 
                 CAGCAAGCACCAAATCACTG 
                 37028305 
                 37028326 
               
               
                   
                   
                   
                 reverse 
                 94 
                 AGTACCAGCCGTCCAAACTG 
                 37032621 
                 37032640 
               
               
                 P3 
                 P3a_MLH1-v1 
                 320 
                 forward 
                 95 
                 CCTGGCCAGAAAATTCATTG 
                 37037607 
                 37037628 
               
               
                   
                   
                   
                 reverse 
                 96 
                 ACCCTGCATTCCAAACTCAC 
                 37039199 
                 37039218 
               
               
                   
                 P3b_MLH1-v1 
                 321 
                 forward 
                 97 
                 GCAGTCCTTTGAGGATTTAGC 
                 37042493 
                 37042515 
               
               
                   
                   
                   
                 reverse 
                 98 
                 GAAAGATATCCAACAGGAAGTGAG 
                 37043300 
                 37043323 
               
               
                   
                 P3c_MLH1-v1 
                 322 
                 forward 
                 99 
                 TGGCCTTGTTTAAGGTCCTG 
                 37043746 
                 37043767 
               
               
                   
                   
                   
                 reverse 
                 100 
                 ATGGTCCTGCTGCTTCAGAG 
                 37044723 
                 37044742 
               
               
                   
                 P3d_MLH1-v1 
                 323 
                 forward 
                 101 
                 ACCCCGTCATAGCACAGTTC 
                 37045295 
                 37045316 
               
               
                   
                   
                   
                 reverse 
                 102 
                 CAAAGGCCATTCATCAGTTTC 
                 37046439 
                 37046459 
               
               
                 P4 
                 P4a_MLH1-v1 
                 324 
                 forward 
                 103 
                 GTGGCGTGATATCCTTGATTC 
                 37053034 
                 37053056 
               
               
                   
                   
                   
                 reverse 
                 104 
                 CTCTGGAATGACTGCTGCTG 
                 37054289 
                 37054308 
               
               
                   
                 P4b_MLH1-v1 
                 325 
                 forward 
                 105 
                 TGTGCTAGATGCCTCACTGG 
                 37055182 
                 37055203 
               
               
                   
                   
                   
                 reverse 
                 106 
                 TTGCCAAGAAGCACAACAAG 
                 37058326 
                 37058345 
               
               
                 P5 
                 P5a1_MLH1-v1 
                 326 
                 forward 
                 107 
                 CGGAGGCTCTACTGTTGGAC 
                 37062345 
                 37062366 
               
               
                   
                   
                   
                 reverse 
                 108 
                 TGCTGTCCACTCTGGAACTG 
                 37064753 
                 37064772 
               
               
                   
                 P5b_MLH1-v1 
                 327 
                 forward 
                 109 
                 ACATCAGAAGCCCTGGTTTG 
                 37064571 
                 37064592 
               
               
                   
                   
                   
                 reverse 
                 110 
                 GCTGGGAGTTCAAGCATCTC 
                 37067377 
                 37067396 
               
               
                 P6 
                 P6a_MLH1-v1 
                 328 
                 forward 
                 111 
                 TCGGTCTCAGTCACCATTTG 
                 37072097 
                 37072118 
               
               
                   
                   
                   
                 reverse 
                 112 
                 AACGCACCTGGCTGAAATAC 
                 37075920 
                 37075939 
               
               
                 P7 
                 P7a_MLH1-v1 
                 329 
                 forward 
                 113 
                 TGAACCTGCAATATCTCAGAGG 
                 37079607 
                 37079630 
               
               
                   
                   
                   
                 reverse 
                 114 
                 CTTACCGATAACCTGAGAACACC 
                 37083805 
                 37083827 
               
               
                 P8 
                 P8a_MLH1-v1 
                 330 
                 forward 
                 115 
                 CCCAGCCCATATATTTTAAAGC 
                 37088387 
                 37088410 
               
               
                   
                   
                   
                 reverse 
                 116 
                 CCAGCCACTCTCTGGACTATC 
                 37089049 
                 37089069 
               
               
                   
                 P8b_MLH1-v1 
                 331 
                 forward 
                 117 
                 GACATGGAGAGCCGAATCC 
                 37089669 
                 37089689 
               
               
                   
                   
                   
                 reverse 
                 118 
                 CCATTAAAATCGGGTCTGAAAG 
                 37091446 
                 37091467 
               
               
                   
                 P8c_MLH1-v1 
                 332 
                 forward 
                 119 
                 TCCAGACCCAGTGCACATC 
                 37091887 
                 37091907 
               
               
                   
                   
                   
                 reverse 
                 120 
                 CATGGTCAGTGCCATCAGAG 
                 37092412 
                 37092431 
               
               
                   
                 P8d_MLH1-v1 
                 333 
                 forward 
                 121 
                 AGCCTCCCAAAGTTAAGTGC 
                 37092788 
                 37092809 
               
               
                   
                   
                   
                 reverse 
                 122 
                 CCCAGCTAAAACCAACACAC 
                 37093346 
                 37093365 
               
               
                 P9 
                 P9a_MLH1-v1 
                 334 
                 forward 
                 123 
                 TGCCCTCAGCTACTCACTCC 
                 37103285 
                 37103306 
               
               
                   
                   
                   
                 reverse 
                 124 
                 AGGGCTCAGCCTTTAGGAAC 
                 37105620 
                 37105639 
               
               
                   
                 P9b_MLH1-v1 
                 335 
                 forward 
                 125 
                 GCCAGACTCTCGTTCCATTC 
                 37106390 
                 37106411 
               
               
                   
                   
                   
                 reverse 
                 126 
                 ACTCCCCATTCAGTCCCTTC 
                 37111053 
                 37111072 
               
               
                   
                 P9c_MLH1-v1 
                 336 
                 forward 
                 127 
                 AGGCACAACGTCAGGTTTTC 
                 37114109 
                 37114130 
               
               
                   
                   
                   
                 reverse 
                 128 
                 TTGGAATTTGTCCTGGTGTG 
                 37117519 
                 37117538 
               
               
                 P10 
                 P10a_MLH1-v1 
                 337 
                 forward 
                 129 
                 CACCATTGCCAACACTTCTG 
                 37132898 
                 37132919 
               
               
                   
                   
                   
                 reverse 
                 130 
                 GCCATTGGTTTGAAGGTGAC 
                 37134201 
                 37134220 
               
               
                   
                 P10b_MLH1-v1 
                 338 
                 forward 
                 131 
                 CTTAGTCACCGCCTGTCCTC 
                 37134738 
                 37134759 
               
               
                   
                   
                   
                 reverse 
                 132 
                 TAGCTGCATGTGGCTAATCG 
                 37136986 
                 37137005 
               
               
                   
                 P10c_MLH1-v1 
                 339 
                 forward 
                 133 
                 TGTGGCTCGCATTACATTTC 
                 37137579 
                 37137600 
               
               
                   
                   
                   
                 reverse 
                 134 
                 CGCTGTCATTACCTGCTTTG 
                 37139742 
                 37139761 
               
               
                   
                 P10d_MLH1-v1 
                 340 
                 forward 
                 135 
                 TGACCTCCAAAATCATCCAG 
                 37140449 
                 37140470 
               
               
                   
                   
                   
                 reverse 
                 136 
                 TTCTGAGCTAGGAGGTGCTG 
                 37141321 
                 37141340 
               
               
                   
                 P10e_MLH1-v1 
                 341 
                 forward 
                 137 
                 CCAGATTTGTAAATCCCTGTTC  
                 37142008 
                 37142031 
               
               
                   
                   
                   
                 reverse 
                 138 
                 TGTGTGGTTCTTAAGCATTCC 
                 37142420 
                 37142440 
               
             
          
           
               
                 MSH2-v2 
               
             
          
           
               
                 tPP1 
                 tPP1a_MSH2-v2 
                 342 
                 forward 
                 139 
                 CTCAGTCCATCAGCCTCCTC 
                 47574824 
                 47577784 
               
               
                   
                   
                   
                 reverse 
                 140 
                 TGCTGTGCCCTGAGATTAAG 
                 47574823 
                 47577783 
               
               
                   
                 tPP1b_MSH2-v2 
                 343 
                 forward 
                 141 
                 AACTTAATCTCAGGGCACAGC 
                 47577763 
                 47580677 
               
               
                   
                   
                   
                 reverse 
                 142 
                 TGCAGCTTCAGCCTCTTG 
                 47577762 
                 47580676 
               
               
                   
                 tPP1c_MSH2-v2 
                 344 
                 forward 
                 143 
                 GCGTGGTGTTTCGTACCAG 
                 47580604 
                 47583785 
               
               
                   
                   
                   
                 reverse 
                 144 
                 GCTACTGGCCAGAAATCTTCC 
                 47580603 
                 47583784 
               
               
                   
                 tPP1d_MSH2-v2 
                 345 
                 forward 
                 145 
                 GCCCAGCCCTACTAAGGAAG 
                 47583750 
                 47586723 
               
               
                   
                   
                   
                 reverse 
                 146 
                 CTGTGCTCCCCTGCTAGAAC 
                 47583749 
                 47586722 
               
               
                   
                 tPP1e_MSH2-v2 
                 346 
                 forward 
                 147 
                 GTCGTCCTCTTCGACCTAGC 
                 47586769 
                 47589967 
               
               
                   
                   
                   
                 reverse 
                 148 
                 CAGCGCCTATTCTACAGCAG 
                 47586768 
                 47589966 
               
               
                 EPCAM5′ 
                 EPCa_MSH2-v2 
                 347 
                 forward 
                 149 
                 TTCTTCCCAAGAGAGCCAAG 
                 47595912 
                 47598965 
               
               
                   
                   
                   
                 reverse 
                 150 
                 CCACCTTTAATCTGCCCAAC 
                 47595911 
                 47598964 
               
               
                   
                 EPCb_MSH2-v2 
                 348 
                 forward 
                 151 
                 GTGTTGGGCAGATTAAAGGTG 
                 47598944 
                 47602122 
               
               
                   
                   
                   
                 reverse 
                 152 
                 GCAGTGTCATGCCCTGTTC 
                 47598943 
                 47602121 
               
               
                   
                 EPCc_MSH2-v2 
                 349 
                 forward 
                 153 
                 CTCTTTGTGCCCTTTCTTTTG 
                 47601745 
                 47604931 
               
               
                   
                   
                   
                 reverse 
                 154 
                 AGTTCCTTAAAGCAGAGAAGATGG 
                 47601744 
                 47604930 
               
               
                 EPCAM3′ 
                 EPCd_MSH2-v2 
                 350 
                 forward 
                 155 
                 AACCTGTCCCTGTGGATGAG 
                 47604796 
                 47607923 
               
               
                   
                   
                   
                 reverse 
                 156 
                 CCGAAGCATCCTTACATTCC 
                 47604795 
                 47607922 
               
               
                   
                 EPCe_MSH2-v2 
                 351 
                 forward 
                 157 
                 AATACCTGAACCCCCAAACC 
                 47607722 
                 47609876 
               
               
                   
                   
                   
                 reverse 
                 158 
                 CTCAGGCTATTTTCCAGATTCAC 
                 47607721 
                 47609875 
               
               
                   
                 EPCf_MSH2-v2 
                 352 
                 forward 
                 159 
                 GCATGCCTGTCATTCTGG 
                 47609695 
                 47612812 
               
               
                   
                   
                   
                 reverse 
                 160 
                 TCCAAGGGACTGAAACACAC 
                 47609694 
                 47612811 
               
               
                   
                 EPCg_MSH2-v2 
                 353 
                 forward 
                 161 
                 TTAGTGTGTTTCAGTCCCTTGG 
                 47612790 
                 47615135 
               
               
                   
                   
                   
                 reverse 
                 162 
                 GACAGCAAGACCAACCCTTC 
                 47612789 
                 47615134 
               
               
                 PE1-2 
                 E1_MSH2-v2 
                 354 
                 forward 
                 163 
                 GCACATTACGAGCTCAGTGC 
                 47629942 
                 47633045 
               
               
                   
                   
                   
                 reverse 
                 164 
                 CTACCAGGAGAACAGCACAGG 
                 47629941 
                 47633044 
               
               
                   
                 E2_MSH2-v2 
                 355 
                 forward 
                 165 
                 TGGGTTAGCATTGTGTTAGGTG 
                 47632899 
                 47636029 
               
               
                   
                   
                   
                 reverse 
                 166 
                 CCACAGGTGTGTGCCAATAG 
                 47632898 
                 47636028 
               
               
                 PE3-6 
                 E3_MSH2-v2 
                 356 
                 forward 
                 167 
                 AAGTTGCAGTTTGGCTGGTC 
                 47635845 
                 47638929 
               
               
                   
                   
                   
                 reverse 
                 168 
                 TTATCTCCAGCGGTGCTTATG 
                 47635844 
                 47638928 
               
               
                   
                 E4_MSH2-v2 
                 357 
                 forward 
                 169 
                 TACCATAAGCACCGCTGGAG 
                 47638906 
                 47642053 
               
               
                   
                   
                   
                 reverse 
                 170 
                 ACTCCACCAAGCCCAGTCTC 
                 47638905 
                 47642052 
               
               
                   
                 E5-6_MSH2-v2 
                 358 
                 forward 
                 171 
                 TTTAGAGACTGGGCTTGGTG 
                 47642030 
                 47644205 
               
               
                   
                   
                   
                 reverse 
                 172 
                 CTCTTCCCCAACAAACCTG 
                 47642029 
                 47644204 
               
               
                 PE7 
                 I6-7_MSH2-v2 
                 359 
                 forward 
                 173 
                 CCCAGTTTCAAGCGATTAAG 
                 47651443 
                 47654570 
               
               
                   
                   
                   
                 reverse 
                 174 
                 AGGAAAAGCATGTTATCTCCAG 
                 47651442 
                 47654569 
               
               
                   
                 E7_MSH2-v2 
                 360 
                 forward 
                 175 
                 TTCCGTAGCAGTAGGCATCC 
                 47654026 
                 47657170 
               
               
                   
                   
                   
                 reverse 
                 176 
                 TCACCACCACCAACTTTATGAG 
                 47654025 
                 47657169 
               
               
                   
                 I7-8_MSH2-v2 
                 361 
                 forward 
                 177 
                 TCCCAGATCTTAACCGACTTG 
                 47656956 
                 47660035 
               
               
                   
                   
                   
                 reverse 
                 178 
                 ATGGCGGTTTTGTGGAATAG 
                 47656955 
                 47660034 
               
               
                 PE8 
                 E8_MSH2-v2 
                 362 
                 forward 
                 179 
                 CCCAAACAACAGCATTAGCC 
                 47670887 
                 47673915 
               
               
                   
                   
                   
                 reverse 
                 180 
                 ACATCAGCCTCGGGACAAG 
                 47670886 
                 47673914 
               
               
                   
                 I8-9a_MSH2-v2 
                 363 
                 forward 
                 181 
                 TGAGCCCGTTGAATATAGTGG 
                 47673830 
                 47675514 
               
               
                   
                   
                   
                 reverse 
                 182 
                 AGTTTTCCTAAACGGGATGATG 
                 47673829 
                 47675513 
               
               
                   
                 I8-9b_MSH2-v2 
                 364 
                 forward 
                 183 
                 ATGGGTGTGCACGTGTGTAG 
                 47675368 
                 47678365 
               
               
                   
                   
                   
                 reverse 
                 184 
                 GCCATGTGCAATTGTGAGTC 
                 47675367 
                 47678364 
               
               
                 PE9 
                 E9_MSH2-v2 
                 365 
                 forward 
                 185 
                 CCTTGCATAGTTTGCTTCTGG 
                 47688375 
                 47690450 
               
               
                   
                   
                   
                 reverse 
                 186 
                 ATCATACAAGGGCCTGTTGG 
                 47688374 
                 47690449 
               
               
                   
                 I9-10_MSH2-v2 
                 366 
                 forward 
                 187 
                 AAACAGAAATCGCCCAACAG 
                 47690418 
                 47692377 
               
               
                   
                   
                   
                 reverse 
                 188 
                 TAGAGACCCACCCAGAAACG 
                 47690417 
                 47692376 
               
               
                 PE10 
                 E10_MSH2-v2 
                 367 
                 forward 
                 189 
                 CAGTCCGATTTCGTTTCTGG 
                 47692347 
                 47695506 
               
               
                   
                   
                   
                 reverse 
                 190 
                 CACACCTAGATTTGGCAATGG 
                 47692346 
                 47695505 
               
               
                 PE11 
                 E11_MSH2-v2 
                 368 
                 forward 
                 191 
                 TTCCATTGCCAAATCTAGGTG 
                 47695484 
                 47698468 
               
               
                   
                   
                   
                 reverse 
                 192 
                 GGCCCTAGTGTTTCCTTTCC 
                 47695483 
                 47698467 
               
               
                   
                 I11-12_MSH2-v2 
                 369 
                 forward 
                 193 
                 AAGGAAACACTAGGGCCTACAAC 
                 47698452 
                 47700589 
               
               
                   
                   
                   
                 reverse 
                 194 
                 CCTGGCCTCAGTACACTTTTG 
                 47698451 
                 47700588 
               
               
                 PE12-14 
                 E12_MSH2-v2 
                 370 
                 forward 
                 195 
                 AGGGATTCTCCCCACTTAGC 
                 47700228 
                 47702718 
               
               
                   
                   
                   
                 reverse 
                 196 
                 ATTGGAGGACTGGCTCAAAG 
                 47700227 
                 47702718 
               
               
                   
                 E13-14_MSH2-v2 
                 371 
                 forward 
                 197 
                 GCTTACCTTTGAGCCAGTCC 
                 47702694 
                 47705819 
               
               
                   
                   
                   
                 reverse 
                 198 
                 ACATGTTCCTACCCCCAGAC 
                 47702693 
                 47705818 
               
               
                 PE15-16 
                 E15_MSH2-v2 
                 372 
                 forward 
                 199 
                 TTTCTGCATCAGTTGGTTGC 
                 47706613 
                 47709532 
               
               
                   
                   
                   
                 reverse 
                 200 
                 GCCAAGTTATTGCTGCTTCAG 
                 47706612 
                 47709531 
               
               
                   
                 E16_MSH2-v2 
                 373 
                 forward 
                 201 
                 AGCCCTGTGAGGTTGGTAAC 
                 47709413 
                 47712504 
               
               
                   
                   
                   
                 reverse 
                 202 
                 TCAACAACAGCTGGAACTGC 
                 47709412 
                 47712503 
               
               
                 cPP1 
                 cPP1a_MSH2-v2 
                 374 
                 forward 
                 203 
                 CCTCTCAGGTCAGGCTTCTG 
                 47730898 
                 47733882 
               
               
                   
                   
                   
                 reverse 
                 204 
                 GCTCCCGCTAGAGAAACTCC 
                 47730897 
                 47733881 
               
               
                   
                 cPP1b_MSH2-v2 
                 375 
                 forward 
                 205 
                 GAGCGAAGCACCTAAAGCAC 
                 47733879 
                 47736946 
               
               
                   
                   
                   
                 reverse 
                 206 
                 AATTGGAGGGGGTGGAGTAG 
                 47733878 
                 47736945 
               
               
                   
                 cPP1c_MSH2-v2 
                 376 
                 forward 
                 207 
                 TGTCACCCAGTCAGGTCATC 
                 47736760 
                 47739876 
               
               
                   
                   
                   
                 reverse 
                 208 
                 TTGGAAGGAATCCAACAAGG 
                 47736759 
                 47739875 
               
               
                   
                 cPP1d_MSH2-v2 
                 377 
                 forward 
                 209 
                 TTCCCAGAACTCCTTGTTGG 
                 47739846 
                 47742962 
               
               
                   
                   
                   
                 reverse 
                 210 
                 TGCAAACCCCTTCTTTTCAG 
                 47739845 
                 47742961 
               
               
                   
                 cPP1e_MSH2-v2 
                 378 
                 forward 
                 211 
                 ACCCCATGCAGAAGCAATAG 
                 47743027 
                 47746218 
               
               
                   
                   
                   
                 reverse 
                 212 
                 AAATCCTGAAGGTGGGTTCC 
                 47743026 
                 47746217 
               
             
          
           
               
                 MLH1v2 
               
             
          
           
               
                 tPP1 
                 tPP1b_MLH1-v2 
                 379 
                 forward 
                 213 
                 AGTTTCAGCCATGTTGCAG 
                 37005587 
                 37005605 
               
               
                   
                   
                   
                 reverse 
                 214 
                 TTGGCAAAATTGTGACTGAG 
                 37007511 
                 37007530 
               
               
                   
                 tPP1c_MLH1-v2 
                 380 
                 forward 
                 215 
                 CAGTCACAATTTTGCCAAGG 
                 37007513 
                 37007532 
               
               
                   
                   
                   
                 reverse 
                 216 
                 AGTTCGTGGCATCTAACTATCG 
                 37009688 
                 37009709 
               
               
                   
                 tPP1d_MLH1-v2 
                 381 
                 forward 
                 217 
                 GGTCCATGTGCTCCAAAAAG 
                 37009460 
                 37009479 
               
               
                   
                   
                   
                 reverse 
                 218 
                 TCCAAAACTGGGAACAAACC 
                 37012624 
                 37012643 
               
               
                   
                 tPP1e_MLH1-v2 
                 382 
                 forward 
                 219 
                 TGGTTTGTTCCCAGTTTTGG 
                 37012623 
                 37012642 
               
               
                   
                   
                   
                 reverse 
                 220 
                 TAGTGCACCACAGCCTCAAG 
                 37015706 
                 37015725 
               
               
                   
                 tPP1f_MLH1-v2 
                 383 
                 forward 
                 221 
                 GGATCACTTGAGGCTGTGGT 
                 37015700 
                 37015719 
               
               
                   
                   
                   
                 reverse 
                 222 
                 TCCAACAACTGCTGTGAAGG 
                 37018677 
                 37018696 
               
               
                   
                 tPP1g_MLH1-v2 
                 384 
                 forward 
                 223 
                 CACCACTGACCTTCCCTTCC 
                 37018492 
                 37018511 
               
               
                   
                   
                   
                 reverse 
                 224 
                 GCACAGAAAGACAAATATCACATGC 
                 37020534 
                 37020558 
               
               
                   
                 tPP1h_MLH1-v2 
                 385 
                 forward 
                 225 
                 CTCTTCCTCGTCTCCTCCTG 
                 37020430 
                 37020449 
               
               
                   
                   
                   
                 reverse 
                 226 
                 CCAATTCAATGCAAAACCTG 
                 37022464 
                 37022483 
               
               
                 PE1-2 
                 E1_MLH1-v2 
                 386 
                 forward 
                 227 
                 CGAGCAGCTCTCTCTTCAGG 
                 37034273 
                 37034292 
               
               
                   
                   
                   
                 reverse 
                 228 
                 AGCCTATAAGCACAGACCAACTG 
                 37037250 
                 37037272 
               
               
                   
                 E2_MLH1-v2 
                 387 
                 forward 
                 229 
                 TTCTCTAGCAGTTGGTCTGTGC 
                 37037242 
                 37037263 
               
               
                   
                   
                   
                 reverse 
                 230 
                 ACCCTGCATTCCAAACTCAC 
                 37039199 
                 37039218 
               
               
                 PE3-4 
                 I23_MLH1-v2 
                 388 
                 forward 
                 231 
                 GTTCATTTTGGGGCATGTTC 
                 37039148 
                 37039167 
               
               
                   
                   
                   
                 reverse 
                 232 
                 CTGCAACCTCCTTTGAGACAG 
                 37042218 
                 37042238 
               
               
                   
                 E3_MLH1-v2 
                 389 
                 forward 
                 233 
                 TGTCTCAAAGGAGGTTGCAG 
                 37042219 
                 37042238 
               
               
                   
                   
                   
                 reverse 
                 234 
                 CCAAAATGAAACTGCCTTCC 
                 37044367 
                 37044386 
               
               
                   
                 E4_MLH1-v2 
                 390 
                 forward 
                 235 
                 AGTTCCCTGGGTCATTTTCC 
                 37044393 
                 37044412 
               
               
                   
                   
                   
                 reverse 
                 236 
                 TTGTGGGAAGGCAAACTAGC 
                 37046381 
                 37046400 
               
               
                 PE5-6 
                 E5_MLH1-v2 
                 391 
                 forward 
                 237 
                 CCTGTGCTAGTTTGCCTTCC 
                 37046376 
                 37046395 
               
               
                   
                   
                   
                 reverse 
                 238 
                 GGTGGTCACCGTGGTAAAAG 
                 37049553 
                 37049572 
               
               
                   
                 E6_MLH1-v2 
                 392 
                 forward 
                 239 
                 GACCACCATGTGATTTCCAAG 
                 37049566 
                 37049586 
               
               
                   
                   
                   
                 reverse 
                 240 
                 TTGGTTGGCGGTTATTTCTC 
                 37052510 
                 37052529 
               
               
                 PE7-9 
                 E7-8_MLH1-v2 
                 393 
                 forward 
                 241 
                 TAACCGCCAACCAAGAAAAG 
                 37052516 
                 37052535 
               
               
                   
                   
                   
                 reverse 
                 242 
                 TGTCTGGAGACCTTCCCAAG 
                 37055360 
                 37055379 
               
               
                   
                 E9_MLH1-v2 
                 394 
                 forward 
                 243 
                 TGTGCTAGATGCCTCACTGG 
                 37055182 
                 37055201 
               
               
                   
                   
                   
                 reverse 
                 244 
                 ACTTGCCTACATTGCCCATC 
                 37058175 
                 37058194 
               
               
                 PE10-11 
                 E10_MLH1-v2 
                 395 
                 forward 
                 245 
                 ATGGGCAATGTAGGCAAGTC 
                 37058176 
                 37058195 
               
               
                   
                   
                   
                 reverse 
                 246 
                 TCTGCAGCCATGAATAAGTCC 
                 37061070 
                 37061090 
               
               
                   
                 E11_MLH1-v2 
                 396 
                 forward 
                 247 
                 CAGAGCTGAGGCGATAAATTG 
                 37060960 
                 37060980 
               
               
                   
                   
                   
                 reverse 
                 248 
                 TGCTCCTCTCCAATCCATTC 
                 37063973 
                 37063992 
               
               
                 PE12-13 
                 E12_MLH1-v2 
                 397 
                 forward 
                 249 
                 ATACTTTCCCAGCCCAAACC 
                 37066434 
                 37066453 
               
               
                   
                   
                   
                 reverse 
                 250 
                 TGATGGGGAAATGAGAGGAG 
                 37069438 
                 37069457 
               
               
                   
                 E13_MLH1-v2 
                 398 
                 forward 
                 251 
                 AGTGGCCTTTGTCCATTGAG 
                 37069405 
                 37069424 
               
               
                   
                   
                   
                 reverse 
                 252 
                 GACAGAGGTGAGAGCCTAGGAG 
                 37071540 
                 37071561 
               
               
                 PE14-15 
                 E14-15_MLH1-v2 
                 399 
                 forward 
                 253 
                 AATGTGTTGGGGAAGTGGTC 
                 37081262 
                 37081281 
               
               
                   
                   
                   
                 reverse 
                 254 
                 TTTGGACCACGGCTTTAGAC 
                 37084405 
                 37084424 
               
               
                 PE16-19 
                 E16-18_MLH1-v2 
                 400 
                 forward 
                 255 
                 AAGCTGAGGTCACGGATTTG 
                 37087522 
                 37087541 
               
               
                   
                   
                   
                 reverse 
                 256 
                 GATGGGCAAGTTTCATCTCC 
                 37090568 
                 37090587 
               
               
                   
                 E19_MLH1-v2 
                 401 
                 forward 
                 257 
                 TGGGACGAAGAAAAGGAATG 
                 37090401 
                 37090420 
               
               
                   
                   
                   
                 reverse 
                 258 
                 CACCGTGCCTCAGCCTATAC 
                 37093446 
                 37093465 
               
               
                 cPP1 
                 cPP1a_MLH1-v2 
                 402 
                 forward 
                 259 
                 GGACTAACCCACCTCCCTTC 
                 37103239 
                 37103258 
               
               
                   
                   
                   
                 reverse 
                 260 
                 GCTATAGGCAGCCCAGAGTG 
                 37106372 
                 37106391 
               
               
                   
                 cPP2a_MLH1-v2 
                 403 
                 forward 
                 261 
                 GCCAGACTCTCGTTCCATTC 
                 37106390 
                 37106409 
               
               
                   
                   
                   
                 reverse 
                 262 
                 AGGATTTGCCGTATGGACTC 
                 37109450 
                 37109469 
               
               
                   
                 cPP3a_MLH1-v2 
                 404 
                 forward 
                 263 
                 TCGCCCAAAGTCACAGTAAG 
                 37109303 
                 37109322 
               
               
                   
                   
                   
                 reverse 
                 264 
                 GATCTGTAGGCCCAGGATTTC 
                 37112356 
                 37112376 
               
               
                   
                 cPP4a_MLH1-v2 
                 405 
                 forward 
                 265 
                 AGGGGTTTCTATGGCTGGTC 
                 37112314 
                 37112333 
               
               
                   
                   
                   
                 reverse 
                 266 
                 CCTCCCTCAAACCTCCTCTC 
                 37114423 
                 37114442 
               
               
                   
                 cPP5a_MLH1-v2 
                 406 
                 forward 
                 267 
                 TTCTCCTGCAGAGGAAGAGG 
                 37114369 
                 37114388 
               
               
                   
                   
                   
                 reverse 
                 268 
                 TTGGAATTTGTCCTGGTGTG 
                 37117519 
                 37117538 
               
               
                   
                 cPP6a_MLH1-v2 
                 407 
                 forward 
                 269 
                 AAAGCCAGGGAGTGAATGG 
                 37117566 
                 37117584 
               
               
                   
                   
                   
                 reverse 
                 270 
                 ATGTGCATCTCCCTGGTGAC 
                 37120703 
                 37120722 
               
               
                   
                 cPP7a_MLH1-v2 
                 408 
                 forward 
                 271 
                 TGTGGGGAAATCAAAACCTG 
                 37120784 
                 37120803 
               
               
                   
                   
                   
                 reverse 
                 272 
                 GGGTAGACTGTGCGTGTGTG 
                 37123930 
                 37123949 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 MLH1-v2 
                 MLH1-v1 
                 MLH1 
                 MSH2-V2 
                 MSH2-V1 
                 MSH2 
                   
               
               
                   
                 probe 
                 probe 
                 region 
                 probe 
                 probe 
                 region 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 sum length 
                 86366 
                 55582 
                 121536 
                 106534 
                 73609 
                 171394 
                 bp 
               
               
                 repeat 
                 44684 
                 18525 
                 64712 
                 53243 
                 22133 
                 94584 
                 bp 
               
               
                 length 
               
               
                 total repeat 
                 51.74 
                 33.33 
                 53.25 
                 49.98 
                 30.07 
                 55.19 
                 % 
               
               
                 SINE 
                 24.93 
                 2.58 
                 23.85 
                 34.68 
                 5.03 
                 35.95 
                 % 
               
               
                 ALUs 
                 22.38 
                 0.09 
                 21.85 
                 32.85 
                 0.76 
                 34.15 
                 % 
               
               
                   
               
             
          
         
       
     
     REFERENCES 
     
         
         1. “Gene copy number variation and common human disease”, Fanciulli, et. al.  Clinical Genetics,  2010 77, 201-213 
         2. “Dynamic molecular combing: stretching the whole human genome for high-resolution studies” Michalet, et al.,  Science  1997 277, 1518-1523 and “Bar code screening on combed DNA for large rearrangemens of the BRCA1 and BRCA2 gene in French breast cancer families”, Gad, et. al.,  J. Medical Genetics,  2002, 39, 817-821 
         3. “Sequence-based design of single-copy genomic DNA probes for fluorescence in situ hybridization” Rogan, et. al., Genome Res. 200111, 1086-94. 
         4. “A dot-matrix program with dynamic threshold control suited for genomic DNA and protein sequence analysis”. Erik L. L. Sonnhammer and Richard Durbin. Gene 1995, 167:GC1-10 
         5. “Microsatellite instability, mismatch repair deficiency and genetic defects in human cancer cell lines”, Boyer J. C., et al.  Cancer Research  1995 55, 6063-6070, 
         6. “Primer3Plus, an enhanced web interface to Primer3”, Untergasser A., et al.  Nucleic Acids Research  2007 35, W71-W74