Abstract:
The invention relates to a method for enriching a target polynucleotide sequence containing a genetic variation said method comprising: (a) providing two primers targeted to said target polynucleotide sequence; (b) providing a target specific xenonucleic acid clamp oligomer specific for a wildtype polynucleotide sequence; (c) generating multiple amplicons using PCR under specific temperature cycling conditions; and (d) detecting said amplicons.

Description:
[0001]    This application claims the priority benefit under 35 U.S.C. section 119 of U.S. Provisional Patent Application No. 62/010,339 entitled “Method For Enrichment Of Target Mutant Polynucleotide Sequences” filed on Jun. 10, 2014; U.S. Provisional Patent Application No. 62/010,357 entitled “Detection Of Multiple Mutations In A Single Tube Using Qclamp Assay Qclamp Mplex” filed on Jun. 10, 2014; and U.S. Provisional Patent Application No. 62/010,359 entitled “Liquid Biopsy” filed on Jun. 10, 2014; which are in their entirety herein incorporated by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to DNA mutation detection. The invention further relates to enrichment of mutant polynucleotide sequences. The present invention further relates to minimally invasive sampling and analysis of mutations in clinical samples. 
         [0003]    The instant invention also relates to a method for determining whether a target polynucleotide sequence contained in a nucleic acid sample has nucleotide variation(s) in a selected region thereof, the steps of which involve the use of a pair of primers that allows the formation of a PCR product having a sequence covering that of the selected region of the target polynucleotide sequence via a PCR process, and a peptide nucleic acid (PNA) that acts as a PCR clamp as well as a sensor probe. This invention also relates to a kit for use in determining the presence of nucleotide variation(s) in the target polynucleotide sequence, which comprises the pair of primers and the PNA. 
         [0004]    The present embodiments relate to precision molecular diagnostics, and in particular, to compositions in detecting sequence variants, such as SNPs, insertions deletions, and altered methylation patterns, from samples. The embodiments disclosed herein can be used to detect (and quantify) sequence variants present in samples that include an excess of wild-type sequences. 
       BACKGROUND OF THE INVENTION 
       [0005]    Polymerase chain reaction (PCR) is a widely used technique for the detection of pathogens. The technique uses a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. The PCR process generates DNA that is used as a template for replication. This results in a chain reaction that exponentially amplifies the DNA template. 
         [0006]    Technologies for genomic detection most commonly use DNA probes to hybridize to target sequences. To achieve required sensitivity, the use of PCR to amplify target sequences has remained standard practice in many labs. While PCR has been the principle method to identify genes associated with disease states, the method has remained confined to use within a laboratory environment. Most current diagnostic applications that can be used outside of the laboratory are based on antibody recognition of protein targets and use ELISA-based technologies to signal the presence of a disease. These methods are fast and fairly robust, but they can lack the specificity associated with nucleic acid detection. 
         [0007]    With the advent of molecular diagnostics and the discovery of numerous nucleic acid biomarkers useful in the diagnosis and treatment of conditions and diseases, detection of nucleic acid sequences, and sequence variants, mutations and polymorphisms has become increasingly important. In many instances, it is desirable to detect sequence variants or mutations (which may in some instances, differ by one a single nucleotide) present in low copy numbers against a high background of wild-type sequences. For example, as more and more somatic mutations are shown to be biomarkers for cancer prognosis and prediction of therapeutic efficacy, the need for efficient and effective methods to detect rare mutations in a sample is becoming more and more critical. In the case in which one or more allelic variants is/are present in low copy number compared to wild-type sequences, the presence of excess wild-type target sequence creates challenges to the detection of the less abundant variant target sequence. Nucleic acid amplification/detection reactions almost always are performed using limiting amounts of reagents. A large excess of wild-type target sequences, thus competes for and consumes limiting reagents. As a result amplification and/or detection of rare mutant or variant alleles under these conditions is substantially suppressed, and the methods may not be sensitive enough to detect the rare variants or mutants. Various methods to overcome this problem have been attempted. These methods are not ideal, however, because they either require the use of a unique primer for each allele, or the performance of an intricate melt-curve analysis. Both of these shortcomings limit the ability and feasibility of multiplex detection of multiple variant alleles from a single sample. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0008]      FIG. 1  illustrates the mechanism of the XNA clamping process. 
           [0009]      FIG. 2  shows the differential melting temperature (Tm) between the XNA clamp bound to mutant templates vs wild type templates. 
           [0010]      FIG. 3  show specific hydrolysis probe having a different fluorophore (and quencher) selected from the available fluorophores for multiplex applications. 
           [0011]      FIG. 4  is a representative fluorophore spectral data and quencher selection guide. 
           [0012]      FIG. 5  shows a specific locus specific hydrolysis probe assay. 
           [0013]      FIG. 6  is a schematic illustrating how circulating tumor cells (CTC&#39;s) and cell-free DNA (cfDNA) derived from tumor cells are present in the peripheral blood of cancer patients 
       
    
    
     SUMMARY OF THE INVENTION 
       [0014]    Detection of rare sequence variants in biological samples presents numerous challenges. The methods and kits disclosed herein provide for improved, efficient means to detect rare mutations within a high background of wild-type allelic sequences using real-time amplification methods. 
         [0015]    The instant invention provides a method for enriching a target polynucleotide sequence containing a genetic variation said method comprising: (a) providing two primers targeted to said target polynucleotide sequence; (b) providing a target specific xenonucleic acid clamp oligomer specific for a wildtype polynucleotide sequence; (c) generating multiple amplicons using PCR under specific temperature cycling conditions; and (d) detecting said amplicons. 
         [0016]    The invention also relates to a method for enriching multiple target polynucleotide sequences containing a genetic variation said method comprising: (a) providing a library of amplifying primers targeted to said multiple target polynucleotide sequence; (b) providing a library of target specific xenonucleic acid clamp oligomer specific for multiple wildtype polynucleotide sequences; (c) generating multiple amplicons using PCR under specific temperature cycling conditions; and (d) detecting said amplicons. 
         [0017]    The invention further relates to a method for conducting a minimally invasive biopsy in a mammalian subject suspected of a having a neoplastic disease, said method comprising: (a) sampling of target polynucleotides derived from said mammalian subject; (b) providing a library of amplifying primers targeted to said multiple target poly-nucleotide sequence; (c) providing a library of target specific xenonucleic acid clamp oligomer specific for multiple wildtype polynucleotide sequences; (d) generating multiple amplicons using PCR under specific temperature cycling conditions; and (e) detecting said amplicons. 
         [0018]    The invention is also directed to means and methodology for the rapid isolation of genetic material from biological fluids and the sensitive detection of somatic and germ-line mutations present in circulating cells and cell-free genetic material obtained from said biological fluids using gene amplification and xeno-nucleic acid (XNA) clamping. 
         [0019]    This invention provides a method for determining whether a target polynucleotide sequence contained in a nucleic acid sample has nucleotide variation(s) in a selected region thereof, comprising the steps of: providing a pair of a first primer and a second primer which allows the formation of a PCR product having a sequence covering that of the selected region of the target polynucleotide sequence via a PCR process, the first primer having a sequence identical to that of a first region located upstream of the selected region of the target polynucleotide sequence, the second primer having a sequence based on that of a second region located downstream of the selected region of the target polynucleotide sequence, wherein the 6′-end of the sequence of the first region is spaced apart from the 5′-end of the sequence of the sequence of the selected region by 30 nucleotides or more; 
         [0020]    providing a detectable peptide nucleic acid probe having a sequence that complements fully the sequence of the selected region of the target polynucleotide sequence having no nucleotide variation(s) therein, such that hybridization of the detectable peptide nucleic acid probe to the selected region of the target polynucleotide sequence having no nucleotide variation(s) results in the formation of a duplex having a melting temperature; 
         [0021]    determining the melting temperature of the duplex; 
         [0022]    admixing the detectable peptide nucleic acid probe and the pair of the first primer and the second primer with the nucleic acid sample to form a mixture; 
         [0023]    subjecting the mixture to a PCR process including an extension reaction set to run at a temperature lower than the melting temperature of the duplex by 5 to 20° C., such that a mixture of PCR products is obtained; and 
         [0024]    subjecting the mixture of PCR products thus-obtained to a melting analysis to determine melting temperatures of the PCR products, wherein the presence of at least one melting temperature lower than the melting temperature of the duplex is indicative of the nucleotide variation(s) in the selected region of the target polynucleotide sequence contained in the nucleic acid sample. 
         [0025]    The invention also provides a kit for determining whether a target polynucleotide sequence contained in a nucleic acid sample has nucleotide variation(s) in a selected region thereof, comprising: a detectable peptide nucleic acid probe having a sequence that complements fully the sequence of the selected region of the target polynucleotide sequence having no nucleotide variation(s) therein, such that hybridization of the detectable peptide nucleic acid probe to the selected region of the target polynucleotide sequence having no nucleotide variation(s) results in the formation of a duplex having a melting temperature; 
         [0026]    a pair of a first primer and a second primer which allows the formation of a PCR product having a sequence covering that of the selected region of the target polynucleotide sequence via a PCR process, the first primer having a sequence identical to that of a first region located upstream of the selected region of the target polynucleotide sequence, the second primer having a sequence based on that of a second region located downstream of the selected region of the target polynucleotide sequence, wherein the 5′-end of the sequence of the first region is spaced apart from the 5′-end of the sequence of the sequence of the selected region by 30 nucleotides or more; and an instruction sheet providing guidance for a user to use the detectable peptide nucleic acid probe and the pair of the first primer and the second primer in a method as described above. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0027]    It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the current teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of “comprise”, “contain”, and “include”, or modifications of those root words, for example but not limited to, “comprises”, “contained”, and “including”, are not intended to be limiting. Use of “or” means “and/or” unless stated otherwise. The term “and/or” means that the terms before and after can be taken together or separately. For illustration purposes, but not as a limitation, “X and/or Y” can mean “X” or “Y” or “X and Y”. Whenever a range of values is provided herein, the range is meant to include the starting value and the ending value and any value or value range there between unless otherwise specifically stated. For example, “from 0.2 to 0.5” means 0.2, 0.3, 0.4, 0.5; ranges there between such as 0.2-0.3, 0.3-0.4, 0.2-0.4; increments there between such as 0.25, 0.35, 0.225, 0.335, 0.49; increment ranges there between such as 0.26-0.39; and the like. 
         [0028]    In a first embodiment, the present invention relates to compositions and methods for the selective enrichment of low-abundance polynucleotides in a sample. These methods use xeno-nucleic acid (XNA) nucleobase oligomers to selectively block DNA polymerase activity on high abundance wild-type DNA templates, thereby resulting in enrichment of less abundant mutated DNA templates present in a biological sample during a polymerase chain reaction (PCR). The methodology of the present invention can be used to improve DNA sequencing (Sanger sequencing and Pyrosequencing) and also enhance cDNA library preparation for next generation DNA sequencing (NGS). 
         [0029]    Utilizing xeno-nucleic acid (XNA) clamping probes in the PCR mediated amplification of DNA templates, only target genetic material that has a variation, e.g. single nucleotide polymorphism (SNP), gene deletion or insertion and/or translocation or truncation is amplified in the oligonucleotide primer directed polymerase chain reaction (qPCR). 
         [0030]    The XNA probe clamping sequences are designed to bind specifically by Watson-Crick base pairing to abundant wild-type sequences in the DNA templates derived from the biological sample of interest. The presence of the XNA probes in the PCR primer mix employed for the target amplification reaction causes inhibition of the polymerase mediated amplification of wild-type templates but does not impede the amplification of mutant template sequences. 
         [0031]    The mechanism of the XNA clamping process is depicted in  FIG. 1 . As shown in  FIG. 1 , the modified DNA oligo probe binds or clamps to wild type DNA and blocks further wild type amplification. This probe or XNA “clamp” does not bind to mutated DNA, allowing it to be amplified and detected. 
         [0032]    The suppression of wild-type (wt) template amplification and amplification of only mutant templates is achieved because there is a differential melting temperature (Tm) between the XNA clamp bound to mutant templates vs wild type templates: 
         [0000]      Tm(XNA mutant template)&lt;&lt;Tm(XNA wt template) 
         [0033]    The Tm differential is as much as 15-20° C. for the XNA clamp probes. So that during the PCR process only mutant templates are amplified. 
         [0034]    The methods disclosed herein can be used to analyze nucleic acids of samples. The term “sample” as described herein can include bodily fluids (including, but not limited to, blood, urine, feces, serum, lymph, saliva, anal and vaginal secretions, perspiration, peritoneal fluid, pleural fluid, effusions, ascites, and purulent secretions, lavage fluids, drained fluids, brush cytology specimens, biopsy tissue (e.g., tumor samples), explanted medical devices, infected catheters, pus, biofilms and semen) of virtually any organism, with mammalian samples, particularly human samples. 
         [0035]    Amplification primers useful in the embodiments disclosed herein are preferably between 10 and 45 nucleotides in length. For example, the primers can be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more nucleotides in length. Primers can be provided in any suitable form, included bound to a solid support, liquid, and lyophilized, for example. In some embodiments, the primers and/or probes include oligonucleotides that hybridize to a reference nucleic acid sequence over the entire length of the oligonucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other. Where an oligonucleotide is referred to as “substantially complementary” with respect to a nucleic acid sequence herein, the two sequences can be fully complementary, or they may form mismatches upon hybridization, but retain the ability to hybridize under stringent conditions or standard PCR conditions as discussed below. As used herein, the term “standard PCR conditions” include, for example, any of the PCR conditions disclosed herein, or known in the art, as described in, for example, PCR 1: A Practical Approach, M. J. McPherson, P. Quirke, and G. R. Taylor, Ed., (c) 2001, Oxford University Press, Oxford, England, and PCR Protocols: Current Methods and Applications, B. White, Ed., (c) 1993, Humana Press, Totowa, N.J. The amplification primers can be substantially complementary to their annealing region, comprising the specific variant target sequence(s) or the wild type target sequence(s). Accordingly, substantially complementary sequences can refer to sequences ranging in percent identity from 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 85, 80, 75 or less, or any number in between, compared to the reference sequence. Conditions for enhancing the stringency of amplification reactions and suitable in the embodiments disclosed herein, are well-known to those in the art. A discussion of PCR conditions, and stringency of PCR, can be found, for example in Roux, K. “Optimization and Troubleshooting in PCR,” in Pcr Primer: A Laboratory Manual, Diffenbach, Ed. © 1995, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Datta, et al. (2003) Nucl. Acids Res. 31(19):5590-5597. 
         [0036]    Provided herein are methods useful in the detection of sequence variants, i.e., insertions, deletions, nonsense mutations, missense mutations, and the like. In the methods for detecting allelic variants or variant target sequences disclosed herein, the sample, which comprises the nucleic acids to be analyzed, are contacted with an amplification primer pair, i.e., comprising a forward primer and a reverse primer that flank the target sequence or target region containing a sequence of interest {e.g., a wild-type, mutant, or variant allele sequence) to be analyzed. By “flanking” the target sequence, it is understood that the variant or wild-type allelic sequence is located between the forward and reverse primers, and that the binding site of neither the forward nor reverse primer comprises the variant or wild-type allelic sequence to be assessed. For example, in some embodiments, the variant or wild-type allelic sequence to be assessed is removed from or positioned away from the 3′ end of either oligonucleotide by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more, e.g., 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, etc., nucleotides. Amplification primers that flank, but that do not overlap with, the variant target sequence or the wild-type target sequence are thus not “allele-specific” amplification primers, and are capable of amplification of various different alleles or variants of a sequence of interest. Thus, in some embodiments, the amplification primers are configured to amplify various mutant or variant alleles and wild type alleles non-preferentially. As discussed in further detail below, the addition of XNA or PNA to an amplification reaction suppresses the amplification of wild-type target sequences and enables preferential amplification of non-wild-type, e.g., variant, mutant or rare variant alleles.  FIG. 1  is a depictions of exemplary method according to the embodiments disclosed herein for the detection of sequence variants. As shown in  FIG. 1 , amplification primers (i.e., forward primer 1 and reverse primer 2) flank the wild type and mutant allele sequences of interest, and comprise sequences common to both wild-type and mutant or variant allele sequences. Accordingly, as shown in  FIG. 1 , in contrast to methods that utilize allele-specific amplification primers to achieve preferential amplification of rare sequences, the present methods advantageously enable the simultaneous amplification of multiple variant sequences, using a single amplification primer pair. 
         [0037]    In a second embodiment, the invention relates to compositions and methods for the detection of genetic variations (mutations) in DNA templates derived from biological samples with xeno-nucleic acid clamping probes. The first method employs multi-color fluorescence detection using locus specific fluorescent hybridization probes (Hyb Probes), hydrolysis (TaqMan or ZEN) probes or molecular beacons. The second method employs mutant specific amplicon capture probes immobilized on multiple bar-coded capture beads. 
         [0038]    Current XNA clamping qPCR methodologies utilize a single tube—single mutation detection format it is preferable to detect multiple genetic variations in a single tube thus reducing the complexity of the assay and the amount of template DNA required for analysis. 
         [0039]    This second embodiment of the invention is directed to the use of locus specific fluorescent probes designed to detect the genetic variant (mutant) amplicons generated during the XNA clamping PCR reaction. This second embodiment discloses locus specific probes that bind to mutant specific amplicons at a region upstream or downstream from the site of the mutation to be detected. Furthermore, the second embodiment discloses the use of multiplexed XNA clamping qPCR reactions that are able to detect multiple mutations (up to a maximum of 6) in one PCR reaction tube using fluorescence detection methodology. 
         [0040]    In a third embodiment of the invention, there is provided a method the rapid isolation of genetic material present in circulating cells and also cell-free genetic material from biological fluids and the determination of genetic variations in those cells and biological fluids. Such biological fluids include: blood, serum, plasma, saliva, mucus, urine, sputum, semen or other biological secretions. In this embodiment, the invention also provides the detection of somatic and germ-line mutations in the genetic material derived from these biological fluids utilizing gene amplification and xeno-nucleic acid clamping. 
         [0041]    Circulating tumor cells (CTC&#39;s) and cell-free DNA (cfDNA) derived from tumor cells are present in the peripheral blood of cancer patients (See  FIG. 6 ). Tumor derived DNA can also be found in the urine and even the saliva of cancer patients. 
         [0042]    In general circulating free DNA is smaller in size than DNA derived directly from a surgical biopsy or FFPE sample. This embodiment also describes a novel sample treatment procedure that utilizes a novel lysis reagent called QZol™. QZol™ sample lysis is a direct one tube procedure and an aliquot of the lysate is used directly in molecular genetic and cytogenetic analysis procedures such as PCR, RTPCR, FISH, Next Generation Sequencing (NGS) and branched DNA (bDNA) assays. The QZol™ procedure eliminates the tedious multistep preanalytical processing that is currently used in Molecular Pathology and Cytogenetic analysis. 
         [0043]    The lysis reagent is a 50% solution (A) containing chaotropic salts and detergent (nonionic, anionic, cationic or zwitterionic) and a 50% solution (B) containing neutralizing reagents and stabilizers. 
         [0044]    This invention also concerns to the specific amplification of genetic variant templates from the isolated genetic material described above. Only target genetic material that has a variation, e.g. single nucleotide polymorphism (SNP), gene deletion or insertion and/or translocation or truncation is amplified in a quantitative primer directed polymerase chain reaction (qPCR). This is achieved utilising xenonucleic acid (XNA) probe clamping sequences that have been designed to bind specifically by Watson-Crick base pairing to wild-type sequences in the sample. The presence of the XNA probes in the qPCR primer mix employed for the target amplification reaction causes inhibition of the polymerase mediated amplification of wild-type templates but does not impede the amplification of mutant template sequences. 
         [0045]    The mechanism of the XNA clamping process is depicted in  FIG. 1 . 
         [0046]    The suppression of wild-type (wt) template amplification and amplification of only mutant templates is achieved because there is a differential melting temperature (Tm) between the XNA clamp bound to mutant templates vs wt templates: 
         [0000]      Tm(XNA mutant template)&lt;&lt;Tm(XNA wt template) 
         [0000]    The Tm differential is as much as 15-20° C. for the XNA clamp probes. So that during the qPCR process only mutant templates are amplified. 
         [0047]    The methods disclosed herein can be used in the detection of numerous allelic variants, including nonsense mutations, missense mutations, insertions, deletions, and the like. Owing to the advantageous sensitivity and specificity of detection afforded by the methods disclosed herein, the methods can detect the presence of a rare allelic variant within a sample, amongst a high wild-type background. Accordingly, although the skilled artisan will appreciate that the methods disclosed herein can be used in a variety of settings to detect, e.g., germline mutations, the methods are particularly well-suited for use in the detection of somatic mutations, such as mutations present in tumors. Non-limiting examples of rare, somatic mutations useful in the diagnosis, prognosis, and treatment of various tumors include, for example, mutations in ABL, AKT1, AKT2, ALK, APC, ATM, BRAF, CBL, CDH1, CDK 2A, CEBPA, CRLF2, CSF1R, CTNNB1, EGFR, ERBB2, EZH2, FBXW7, FGFR, FGFR2, FGFR3, FLT3, FOXL2, GATA1, GATA2, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH3, JAK2, KIT, KRAS, MEK1, MET, MPL, NF2, NOTCH 1, NOTCH2, NPM, NRAS, PC A3, PDGFRA, PIK3CA, PIK3R1, PIK3R5, PTCH1, PTEN, PTPN1 1, RBI, RET, RUNX1, SMAD4, SMARCB, SMO, STK11, TET2, P53, TSHR, VHL, WT1, and others. Exemplary mutant alleles associated with cancer useful in the embodiments disclosed herein include, but are not limited to those described in publications listed on the world wide web site for COSMIC (Catalogue Of Somatic Mutations In Cancer). 
       EXAMPLES 
     Example 1 
       [0048]    The kit described in great detail in this Example is a KRAS mutation detection kit. However, the same type of kit may be assembled to detect mutations in NRAS, EGFR, BRAF, PIK3CA, JAK2, as well as other genes of importance in precision molecular diagnostics. 
       QCLAMP™ Technology for Mutation Detection 
       [0049]    The QClamp™ KRAS Mutation Detection Kit is based on xenonucleic acid (XNA) mediated PCR clamping technology. XNA is a synthetic DNA analog in which the phosphodiester backbone has been replaced by a repeat formed by units of (2-aminoethyl)-glycine. XNAs hybridize tightly to complementary DNA target sequences only if the sequence is a complete match. Binding of XNA to its target sequence blocks strand elongation by DNA polymerase. When there is a mutation in the target site, and therefore a mismatch, the XNA:DNA duplex is unstable, allowing strand elongation by DNA polymerase. Addition of an XNA, whose sequence with a complete match to wild-type DNA, to a PCR reaction, blocks amplification of wild-type DNA allowing selective amplification of mutant DNA. XNA oligomers are not recognized by DNA polymerases and cannot be utilized as primers in subsequent real-time PCR reactions. 
       DNA Isolation 
       [0050]    Human genomic DNA must be extracted from tissue or blood, or fixed paraffin-embedded tissue prior to use. Several methods exist for DNA isolation. For consistency, we recommend using a commercial kit, such as Qiagen DNA extraction kit (QIAamp DNA FFPE Tissue Kit, cat No. 56404, for paraffin embedded specimens; DNeasy Blood &amp; Tissue kit, cat. No. 69504 or 69506, for tissue and blood specimens). Follow the genomic DNA isolation procedure according to manufacturer&#39;s protocol. Sufficient amounts of DNA can be isolated from FFPE blocks or fresh frozen sections (approx. 2-10 μg). 
         [0051]    This QClamp assay requires a total of 30-60 ng of DNA per sample (5-10 ng/reaction). After DNA isolation, measure the concentration using spectrophotometric analysis (i.e. Nanodrop or UV spectrophotometer) and dilute to it to 1.25-2.5 ng/μl. Make sure A260/A230 value is greater than 2.0 and A260/A280 value between 1.8 and 2.0. 
       Preparation of Reagents 
       [0052]    Each kit contains enough material to run 3 sets (10-sample test kit) or 6 sets (30-sample test kit) of Clamping Controls, Positive Controls and Non-Template Controls. Thaw all Primers, XNAs, Positive Control, WT Clamping Control, water and 2×PCR Mastermix provided. Thaw all reaction mixes at room temperature for a minimum of 1 hour. Vortex all components except the PCR Master Mix the reaction mixes for 5 sec and perform a quick spin. The PCR Master Mix should be mixed gently by inverting the tube a few times. Do not leave kit components at room temperature for more than 4 hours. After thawing, keep materials on ice at all times. The PCR reactions are set up in a total volume of 20 μl/reaction. 
         [0053]    Table 1 shows the component volumes for each 20 ul reaction. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 QCLAMP Assay Components and Reaction Volume 
               
             
          
           
               
                   
                 Components 
                 Volume/Reaction 
               
               
                   
                   
               
               
                   
                 2X PCR Master mix 
                 10 μl 
               
               
                   
                 Primer Mix 
                  4 μl 
               
               
                   
                 XNA 
                  2 μl 
               
               
                   
                 DNA sample or Controls 
                  4 μl 
               
               
                   
                 Total volume 
                 20 μl 
               
               
                   
                   
               
             
          
         
       
     
         [0054]    For accuracy, 2×PCR Mastermix, primers and XNA should be pre-mixed into assay mixes as described in Table 2 below. 
       Preparation of Assay Mixes 
       [0055]    IMPORTANT: Assay mixes should be prepared just prior to use. Do not store assay mixes. Prepare and keep assay mixes on ice, until ready for per. Label 7 micro centrifuge tubes (not provided) according to each corresponding reaction mix shown in Table 2. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Preparation of Assay Mixes 
               
             
          
           
               
                   
                   
                   
                 Volume  
               
               
                   
                   
                   
                 of XNA 
               
               
                   
                 Volume of 2X PCR 
                 Volume of Primer 
                 (†use water 
               
               
                   
                 Master Mix 
                 Mix 
                 for ext control) 
               
               
                   
               
               
                 Ext Control Mix 
                 10 μl × (*n + 1) 
                 4 μl × (*n + 1) 
                 2 μl × (*n + 1) 
               
               
                 G12 Mix 
                 10 μl × (*n + 1) 
                 4 μl × (*n + 1) 
                 2 μl × (*n + 1) 
               
               
                 G13 Mix 
                 10 μl × (*n + 1) 
                 4 μl × (*n + 1) 
                 2 μl × (*n + 1) 
               
               
                 A59 Mix 
                 10 μl × (*n + 1) 
                 4 μl × (*n + 1) 
                 2 μl × (*n + 1) 
               
               
                 Q61 Mix 
                 10 μl × (*n + 1) 
                 4 μl × (*n + 1) 
                 2 μl × (*n + 1) 
               
               
                 K117 Mix 
                 10 μl × (*n + 1) 
                 4 μl × (*n + 1) 
                 2 μl × (*n + 1) 
               
               
                 A146 Mix 
                 10 μl × (*n + 1) 
                 4 μl × (*n + 1) 
                 2 μl × (*n + 1) 
               
               
                   
               
               
                 *n = number of reactions (DNA samples plus 3 controls). Prepare enough for 1 extra sample (n + 1) to allow for sufficient overage for the PCR set. 
               
               
                 †Use 2 ul of water provided in the kit as the Ext Control Mix does not require XNA. For accuracy, do not pipette less than 10 ul of the XNA. 
               
             
          
         
       
     
         [0056]    Prepare sufficient working assay mixes for the DNA samples, one KRAS Mixed Positive Control, one Nuclease-Free Water for no template control, and one WT Clamping Control, according to the volumes in Table 2. Include reagents for 1 extra sample to allow sufficient overage for the PCR set up. The master mixes contain all of the components needed for PCR except the sample. 
         [0057]    Each sample requires one reaction for each mutation site detected by the kit and an external control. The External Control uses Exon 5 primers to determine if an appropriate level of amplifiable DNA is present in the sample, and ensures that that the supplied primers and polymerase are working properly on the sample. The KRAS Codon-Specific kit requires a total of 7 reactions for each sample. 
         [0058]    A set of clamping controls must be run with each of the 7 reaction mixes, every time the assay is run. Clamping Controls use wild-type DNA as the template. Wild-type DNA should have no mutations, therefore the XNA probes will bind strongly, blocking the polymerase from making amplicons. However, the External Control Mix with the Clamping Control should make amplicons efficiently, providing another way to monitor performance of the primers, polymerase, and sample. 
         [0059]    A set of positive controls must also be run with each of the 7 reaction mixes, every time the assay is run. The Positive Control contains one mutant template for each reaction mix. Positive controls contain mutations; therefore XNA probes will not bind, allowing amplification of the mutant template. Positive controls must show the appropriate values for the reaction to be valid. 
         [0060]    A set of no template control (tube NTC) is run with each of the 7 reaction mixes every time the assay is run. Nuclease-Free Water is used in the place of template. The NTC serves as a negative control and assesses potential contamination during assay set-up. 
         [0061]    Further quantities of KRAS Wild-Type Genomic Reference DNA Control, and Positive Control mixes can be purchased as a separate item, if desired. 
       Suggested Run Layout (96-Well Plate, Tube Strips, or Tubes) 
       [0062]    Gently vortex the assay mixes for 5 sec and do a quick spin. Add 16 μl of the appropriate assay mix to the plate or tubes. Add 4 μl of template. Prepare and keep on ice until ready for PCR. 
         [0063]    In the case of 96-well plates, the exact plate layout can be set to the user&#39;s preference. However, take care to remember which wells are for which reaction mixes, to ensure that all potential detected mutations and controls are processed properly. 
         [0000]    Table 3 is a suggested plate set-up for a single experiment analyzing 3 unknown samples. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Suggested Plate Layout 
               
             
          
           
               
                   
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
               
               
                   
               
               
                 A 
                 NTC 
                 PC 
                 CC 
                 S1 
                 S2 
                 S3 
               
               
                   
                 Ext Ctrl  
                 Ext Ctrl  
                 Ext Ctrl  
                 Ext Ctrl  
                 Ext Ctrl  
                 Ext Ctrl  
               
               
                   
                 Mix 
                 Mix  
                 Mix 
                 Mix 
                 Mix 
                 Mix 
               
               
                 B 
                 NTC 
                 PC 
                 CC 
                 S1 
                 S2 
                 S3 
               
               
                   
                 G12  
                 G12  
                 G12  
                 G12 Mix 
                 G12 Mix 
                 G12 Mix 
               
               
                   
                 Mix 
                 Mix 
                 Mix 
                   
                   
                   
               
               
                 C 
                 NTC 
                 PC 
                 CC 
                 S1 
                 S2 
                 S3 
               
               
                   
                 G13  
                 G13  
                 G13  
                 G13 Mix 
                 G13 Mix 
                 G13 Mix 
               
               
                   
                 Mix 
                 Mix 
                 Mix 
                   
                   
                   
               
               
                 D 
                 NTC 
                 PC 
                 CC 
                 S1 
                 S2 
                 S3 
               
               
                   
                 A59  
                 A59  
                 A59  
                 A59 Mix 
                 A59 Mix 
                 A59 Mix 
               
               
                   
                 Mix 
                 Mix 
                 Mix 
                   
                   
                   
               
               
                 E 
                 NTC 
                 PC 
                 CC 
                 S1 
                 S2 
                 S3 
               
               
                   
                 Q61  
                 Q61  
                 Q61  
                 Q61 Mix 
                 Q61 Mix 
                 Q61 Mix 
               
               
                   
                 Mix 
                 Mix 
                 Mix 
                   
                   
                   
               
               
                 F 
                 NTC 
                 PC 
                 CC 
                 S1 
                 S2 
                 S3 
               
               
                   
                 K117  
                 K117  
                 K117  
                 K117 Mix 
                 K117 Mix 
                 K117 Mix 
               
               
                   
                 Mix 
                 Mix 
                 Mix 
                   
                   
                   
               
               
                 G 
                 NTC 
                 PC 
                 CC 
                 S1 
                 S2 
                 S3 
               
               
                   
                 A146  
                 A146  
                 A146  
                 A146 Mix 
                 A146 Mix 
                 A146 Mix 
               
               
                   
                 Mix 
                 Mix 
                 Mix 
               
               
                   
               
               
                 PC: Positive Control, 
               
               
                 NTC: No Template Control (water), 
               
               
                 CC: Clamping Control (Wild-type DNA), 
               
               
                 S1-3: Samples 1-3. 
               
               
                 NOTE: 
               
               
                 For setup on the Rotor-Gene Q Platforms, the layout must be changed such that the first well contains Positive Control. 
               
             
          
         
       
     
         [0064]    When all reagents have been loaded, tightly close the PCR tubes or seal the 96-well plate to prevent evaporation. Spin at 2000 rpm for 1 minute to collect all the reagents. Place in the real-time PCR instrument immediately or store on ice until the instrument is ready. 
       Instrument Set-Up 
     Roche LightCycler 96 or RocheLightCycler 480 
       [0065]    1. Select New empty experiment&gt;create
 
2. In the Run Editor&gt;Measurement, choose SYBR Green 1 (470/514) channel on (LC96), SYBR Green 1/HRM Dye on (LC480)
 
3. Set up run profile using parameters in Table 7. Ramp rates for the LC 96 and LC480 should match settings below.
 
4. During the analysis set threshold to Auto.
 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Roche Light Cycler, LC96 and LC480 Parameters 
               
             
          
           
               
                   
                 Temper- 
                 Time 
                   
                   
                   
                   
               
               
                   
                 ature 
                 (Sec- 
                   
                 Ramp 
                   
                 Acquisition 
               
               
                 Step 
                 (° C.) 
                 onds) 
                 Cycles 
                 Rate 
                 Mode 
                 Mode 
               
               
                   
               
             
          
           
               
                 PreIncubation 
                 95 
                 300 
                 1 
                 4.4 
                   
                 None 
               
               
                 Denaturation 
                 95 
                 20 
                   
                 2.2 
                 Standard 
                 None 
               
               
                 XNA 
                 70 
                 40 
                 X40 
                 2.2 
                   
                 None 
               
               
                 Annealing 
                   
                   
                   
                   
                   
                   
               
               
                 Primer 
                 64 
                 30 
                   
                 2.2 
                   
                 None 
               
               
                 Annealing 
                   
                   
                   
                   
                   
                   
               
               
                 Extension 
                 72 
                 30 
                   
                 1.0 
                   
                 Single 
               
               
                 Melting 
                 95 
                 10 
                 1 
                 4.4 
                   
                 None 
               
               
                   
                 65 
                 60 
                   
                 2.2 
                   
                 None 
               
               
                   
                 97 
                 1 
                   
                 0.20 
                   
                 Continuous 
               
               
                   
                   
                   
                   
                   
                   
                 (5 readings/ 
               
               
                   
                   
                   
                   
                   
                   
                 ° C.) 
               
               
                 Cooling 
                 37 
                 30 
                 1 
                 2.2 
                   
                 None 
               
               
                   
               
               
                 * An HRM curve or melt analysis should be run at the end of the PCR reaction. This helps to verify the PCR amplification results and with troubleshooting. 
               
             
          
         
       
     
       Applied Biosystems Platforms 
     1. Select File&gt;New Experiment 
       [0066]    2. Enter an experiment name and select 7500 (96 wells) or as appropriate 
       3. Select Quantitation—Standard Curve 
     4. Select SYBR Green Reagents 
       [0067]    5. Select Standard Ramp Rate if available
 
6. Click on Plate Setup in the left navigation panel
 
7. Select the Assign Targets and Samples tab and assign samples to the wells
 
       8. Select NONE for the Passive Reference Dye 
       [0068]    9. Click on Run Method on the left panel, set reaction volume to 20 ul
 
10. Setup the cycling parameters as shown in the table below
 
11. Add Melt Curve at the end of the Cycling Stage. Use continuous and leave default setting for data collection
 
12. During the analysis set threshold to 0.5 (ABI 7900) and 5000 (ABI 7500).
 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Applied Biosystems Platforms Cycling Parameters 
               
             
          
           
               
                   
                   
                 Time 
                   
                 Data  
               
               
                 Step 
                 Temperature (° C.) 
                 (Seconds) 
                 Cycles 
                 Collection 
               
               
                   
               
             
          
           
               
                 PreIncubation 
                 95 
                 300 
                 1 
                 OFF 
               
               
                 Denaturation 
                 95 
                 20 
                 X40 
                 OFF 
               
               
                 XNA Annealing 
                 70 
                 40 
                   
                 OFF 
               
               
                 Primer Annealing 
                 66 
                 30 
                   
                 OFF 
               
               
                 Extension 
                 72 
                 30 
                   
                 ON 
               
               
                 Melt Curve 
                 Default 
                   
                   
                 Continuous 
               
               
                   
               
             
          
         
       
     
       Rotor-Gene Q Platforms 
       [0069]    In the instrument software version 2.1 and above
 
1. Select File&gt;New, Select Three Step with Melt and click New
 
2. Select 72-Well Rotor, check the Locking Ring Attached box, click Next
 
3. Set Reaction volume to 20 ul, click next
 
4. Set Temperature profile as shown in Table 6.
 
       5. Channel Setup: Select Green Source 470 nm, Detector 510 nm, Gain 7 
       [0070]    a. Click Gain Optimization
 
b. Set Temperature to 70 C
 
c. Perform Optimization before 1st acquisition
 
d. Click optimize acquiring
 
e. In the pop-up box enter
 
i. Target Sample Range 5 FL up to 10 FL
 
ii. Acceptable Gain Range −10 to 10
 
f. Click OK, Click Close, Click Next
 
       6. Start-run 
       [0071]    7. During the analysis set threshold to Auto. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 Rotor-Gene Q Platforms Cycling Parameters 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Hold 
                   
                 95° C. 
                  5 minutes 
                 X1 
                 Not Acquiring 
               
               
                 Cycling 
                 Timed Step 
                 95° C. 
                 20 seconds 
                 X40 
                 Not Acquiring 
               
               
                   
                 Timed Step 
                 70° C. 
                 40 seconds 
                   
                 Not Acquiring 
               
               
                   
                 Timed Step 
                 64° C. 
                 30 seconds 
                   
                 Not Acquiring 
               
               
                   
                 Timed Step 
                 72° C. 
                 40 seconds 
                   
                 Acquiring to 
               
               
                   
                   
                   
                   
                   
                 Cycling A 
               
               
                   
                   
                   
                   
                   
                 on Green 
               
             
          
           
               
                 Melt 
                   
                 Ramp from 65 to 95, rising by 1 degree each step 
                 Acquire to melt 
               
               
                   
                   
                 Wait for 90 sec of pre-melt conditioning on first 
                 A on green 
               
               
                   
                   
                 step 
                   
               
               
                   
                   
                 Wait for 5 seconds for each step afterwards  
                   
               
               
                   
                   
                 Gain Optimization 
                   
               
               
                   
                   
                 Check optimize gain before melt on all tubes 
                   
               
               
                   
                   
                 The gain giving the highest fluorescence less than 
                   
               
               
                   
                   
                 95 will be selected. 
               
               
                   
               
             
          
         
       
     
       Assessment of Real-Time PCR Results 
       [0072]    For the analysis use Absolute Quantitation, automatic baseline. The threshold to be used with each instrument is listed above. Check threshold to ensure that the Threshold is within the exponential growth phase of the amplification plot. If not, the threshold maybe adjusted depending on the run. 
         [0073]    The real-time PCR instrument generates a Cq value. Cq is the cycle threshold, the cycle number at which a signal is detected above background fluorescence. The lower the cycle number at which signal rises above background, the stronger the PCR reaction it represents 
       No Template Controls 
       [0074]    Verify that there is no amplification in no-template controls for each of the reaction mixes. Cq should be undetermined. For some mixes a Cq of 36 or higher may be observed in the NTC. In such cases, check the melting curves obtained. If the melting curve indicates the presence of primer dimers, the reaction may be acceptable. SYBR green binds to primer dimers, resulting in a peak with a lower melting temperature, than the desired amplicon. In many cases formation of primer dimers can be avoided by setting up the PCR reactions on ice, until ready to load into the PCR instrument. 
       Analysis of Clamping and Positive Controls 
       [0075]    The Cq values of the Positive Control (mixed mutant templates) should amplify in the presence of XNAs and yield Cq values given in Table 7. 
         [0000]                                  TABLE 7                   Acceptable Cq Ranges for Positive Controls                    Positive Control               Acceptable Cq Range                       Ext Control   20 ≦ Cq ≦ 26           G12 Mix   ≦32           G13 Mix   ≦32           A59 Mix   ≦32           Q61 Mix   ≦30           K117 Mix   ≦34           A146 Mix   ≦30                       *The Cq value of the Clamping Control (WT DNA) with the Ext Control Mix should be within 20 and 26.           *In addition, the Cq of the Clamping Control with each of the mutation reaction mixes should be at least 3 Cq greater than the Cq of Positive Control with the same reaction mix. If these criteria are not met, the reaction has failed and the results are not valid.           PASS: Cq of Clamping Control with mutation reaction mix—Cq of Positive Control with same mutation reaction mix ≧ 3            
FAIL: Cq of Clamping Control with mutation reaction mix−Cq of Positive Control with same mutation reaction mix ≦3
 
       Judging Validity of Sample Data Based on External Control Mix Results 
       [0076]    The Cq value of the Ext Control Mix can serve as an indication of the purity and the concentration of DNA. Thus, the validity of the test can be decided by the Cq value of the Ext Control Mix. Cq values of any sample with Ext Control Mix should be in the range of 20-27. If the Cq values fall outside the range given in Table 8, the test results should be considered invalid. The experiment should be repeated. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Acceptable Cq Ranges for Samples with External Control Mix 
               
             
          
           
               
                   
                 Cq Value of Ext 
                   
               
               
                 Validity 
                 Control Mix 
                 Descriptions and Recommendations 
               
               
                   
               
               
                 Optimal 
                 20 &lt; Cq &lt; 27 
                 The amplification and amount of DNA sample were optimal. 
               
               
                 Invalid 
                 Cq ≦ 20 
                 Possibility of a false positive is high. Repeat the PCR reaction with 
               
               
                   
                   
                 less DNA. 
               
               
                 Invalid 
                 Cq ≧ 27 
                 Not enough DNA or DNA not pure. The amplification is not 
               
               
                   
                   
                 optimal. Check DNA amount and purity. Repeat the experiment with 
               
               
                   
               
             
          
         
       
     
       Scoring Mutational Status 
       [0077]    IMPORTANT: Refer to the Macro Sheet for QClamp Cq Mutation Analysis for scoring mutational status. Macro maybe requested by contacting information@diacarta.com
 
If a Cq value is undetermined, assign a Cq of 40 and proceed to analysis.
 
The table below should be used to determine mutational status
 
         [0000]                                                      TABLE 9                   Scoring Mutational Status            Mutation       G12   G13   A59   Q61   K117   A146               Strong    Cq   ≦32   ≦32   ≦32   ≦30   ≦33   ≦30       Positive:                                   Mutation                                    Content &gt;                                    5%                                   Weak    Cq   32-35*   32-35*   30-35*   30-35*   33-35*   30-35*       Positive:   ΔCq   ≦10    ≦9    ≦8    ≦8   ≦10    ≦8       Mutation                                    Content                                    1-5%                                   Negative   Cq   ≧35   ≧35   ≦30   ≧35   ≧35   ≧35               *If reaction has been set-up with 5 ng of DNA, it is recommended that the experiment be repeated with 10 ng of template DNA to confirm the results.       *Refer to Table 9 for interpretation of A59/Q61 Mutational Status If the Cq value suggests mutation content between 1%-5%, a further calculation of ΔCq should be performed to determine mutational status.       ΔCq = [Cq value of sample with mutant reaction mix] − [Cq value of sample with Ext Control Mix]        For ex: ΔCq = [Cq of sample with G12 mutant reaction mix] − [Cq of sample with Ext Control Mix]             
Refer to the table above to confirm mutational status of weak positives.
 
       Differentiating A59/Q61 Mutational Status 
       [0078]    The Q61 reaction mix detects both A59 and Q61 mutations, whereas the A59 reaction mix detects only A59 mutations. Therefore, in order to differentiate between A59 and Q61 Mutations a combination of results from the 2 mixes should be used, as described in Table 10 below. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Interpretation of A59/Q61 Mutational Status 
               
             
          
           
               
                 Reaction Mix 
                 Result Based on Table 12  
                 Mutational Status 
               
               
                   
               
               
                 A59 Reaction Mix 
                 Positive 
                 A59 Mutation 
               
               
                 Q61 Reaction Mix 
                 Positive 
                   
               
               
                 A59 Reaction Mix 
                 Negative 
                 Q61 Mutation 
               
               
                 Q61 Reaction Mix 
                 Positive 
                   
               
               
                 A59 Reaction Mix 
                 Negative 
                 Q61 Mutation 
               
               
                 Q61 Reaction Mix 
                 Positive 
               
               
                   
               
             
          
         
       
     
       HRM Curves as a Tool to Confirm Analyses 
       [0079]    In High Resolution Melting Analysis (HRM), the region of interest amplified by PCR is gradually melted. SYBR green is a dsDNA binding dye that is released as the dsDNA amplicon is melted. Emitted fluorescence is measured to generate a characteristic curve. The Tm (Melting Temperature) is characteristic of the GC content, length and sequence of a DNA product and is a useful tool in product identification. The resulting melt profile reflects the mix of amplicons present. 
         [0080]    Wild-type DNA (clamping control) is provided. Some amplification may occur in these reactions. Melt profiles of unknown samples should be compared to wild-type and positive controls. Enrichment of one or more peaks, resulting in a melt profile distinct from wild-type DNA profile, can serve as an indication of specific amplification of a mutation target. If the melt profile of an unknown sample is similar to wild-type DNA, and has been scored as a mutation due to Cq, the analysis should be repeated. The resulting PCR product can be sent for Sanger sequencing for further clarification. 
         [0081]    HRM curves obtained from unknown samples can be compared to HRM curves obtained from positive controls. Amplicons of similar length and sequence will exhibit the same melt profile. 
       Example 2 
       [0082]    PCR based enrichment of mutant DNA template sequences from template DNA derived from a lung cancer tumor biopsy sample is shown below using a xeno-nucleic acid clamping probe specific for KRAS Exon 2 codon 12. Only codon 12 mutant sequences are amplified as shown by the melting profile of the PCR amplicons generated before enrichment and after XNA clamped PCR enrichment: 
         [0083]    The PCR product from the XNA clamped mutant enriched PCR reaction can be isolated and used directly in a Sanger sequencing or Pyrosequencing reaction or else it can be processed for next generation sequencing (NGS) by ligation of adapters and after removal of excess adapters can be used directly for NGS without the need for another PCR amplification step. 
       Example 3 
     Multiplex Detection of KRAS Mutations 
       [0084]    In this example of this invention, locus specific hydrolysis probes are designed to detect mutant amplicons in the KRAS proto-oncogene. Locus specific probes are designed for the following mutant amplicons in KRAS: 
         [0000]    Probe 1 KRAS Exon 2 codon 12,
 
Probe 2 KRAS Exon 2 codon 13,
 
Probe 3 KRAS Exon 3 codon 59
 
Probe 4 KRAS Exon3 codon 61,
 
Probe 5 KRAS Exon 4 codon 117,
 
Probe 6 KRAS Exon 4 codon 146
 
and a control probe for a coding sequence in KRAS that has no mutations—Probe 7 KRAS Control probe
 
         [0085]    Each locus specific hydrolysis probe has a different fluorophore (and quencher) selected from the available fluorophores for multiplex applications (see  FIGS. 3 and 4 ). 
         [0086]    For the KRAS multiplex assay, KRAS c12, c59, c117 and c146 and KRAS control are detected in a one tube and KRAS c13 and c61 and KRAS control in a separate tube. So that all mutations in the KRAS proto-oncogene can be detected using only 2 PCR reaction tubes.  FIG. 5  is an Example of the Exon 4 locus specific probes assay. 
       Example 4 
       [0087]    This example of the invention describes the use of mutation specific capture probes covalently attached to optically bar-coded beads via an amino-linker spacer. Mutant specific probes and control probes for the detection of mutations in KRAS Exon 2 codons 12 and 13 are shown below: 
         [0000]                                         1.   G12A   SEQ ID No: 1   AGCTG   C   TGGCGTA                        2.   G12R   SEQ ID No: 2   AGCT   C   GTGGCGTA                        3.   G12D   SEQ ID No: 3   AGCTG   A   TGGCGTA                        4.   G12C   SEQ ID No: 4   AGCT   T   GTGGCGTA                        5.   G12I   SEQ ID No: 5   GAGCT   AT   TGGCGT                        6.   G12L   SEQ ID No: 6   GAGCT   CT   TGGCGT                        7.   G12S   SEQ ID No: 7   AGCT   A   GTGGCGTA                        8.   G12V   SEQ ID No: 8   AGCTG   T   TGGCGTA                        9.   G13C   SEQ ID No: 9   TGGT   T   GCGTAGGC                       10.   G13D   SEQ ID No: 10   TGGTG   A   CGTAGGC                       11.   G13A   SEQ ID No: 11   TGGTG   C   CGTAGGC                       12.   G13V   SEQ ID No: 12   TGGTG   T   CGTAGGC                       13.   G13S   SEQ ID No: 13   TGGT   A   GCGTAGGC                       14.   G13R   SEQ ID No: 14   TGGT   C   GCGTAGGC            
The control Capture Probes are:
 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 15. 
                 (HLA-)DRA Match 
                 SEQ ID No: 15 
               
               
                   
                   
                 GGAGACGGTCTGG 
                   
               
               
                   
                   
               
               
                   
                 16. 
                 (HLA-)DRA Mismatch 
                 SEQ ID No: 16 
               
               
                   
                   
                 GGAGACG   C   TCTGG 
                   
               
               
                   
                   
               
               
                   
                 17. 
                 KRAS Wild type: 
                 SEQ ID No: 17 
               
               
                   
                   
                 CT   GGTGGC   GTAGG 
                   
               
               
                   
                   
               
               
                   
                 18. 
                 KRAS PCR control 
                 SEQ ID No: 18 
               
               
                   
                   
                 AAGGCCTGCTGAA 
                   
               
             
          
         
       
     
         [0088]    All probes contain a 5′-amino-linker for bar-coded bead conjugation. After, performing XNA clamping PCR reaction is done to eliminate wild-type KRAS using the following primers: KRAS Exon 2 Forward: SEQ ID No: 19 5′-GTACTGGTGGAGTATTTGATAGTG-3′ KRAS Exon 2 Reverse: SEQ ID No: 20 5′-ATCGTCAAGGCACTCTTGCCTAC-3′ and XNA Clamp Probe Blocker specific for KRAS Exon 2 12/13 optically bar-coded mutation specific capture beads are added and incubated for hybridization capture. After washing detection is performed with Streptavidin Phycoerythrin (SAPE) and measured on DigiPlex analyzer. 
       Example 5 
     QClamp Sample DNA Preparation Protocol 
       [0089]    Genomic DNA should be obtained either from whole blood, cells, purified peripheral blood lymphocytes of whole blood, polynuclear cells, or granulocytes, tissue biopsies or FFPE sections. For comparable results it is recommended that the same cellular fraction and DNA extraction method are used. DNA extraction can be performed using a homebrew method or a commercially available kit. 
         [0090]    Carefully transfer FFPE section(s) or equivalent amount of fresh tissue, cells (100 to 100,000 cells) or 200 μl whole blood to a clean 1.7 ml polypropylene micro-centrifuge tube and add the required volume of lysis solution. For FFPE sections add 50 μL of lysis Solution. For liquid or moist cells or tissues add 2× volume of the sample volume. 
         [0091]    For FFPE samples warm each sample in heating block at 95° C. until paraffin melts and then vortex each warm sample for 10 seconds. Return the sealed sample preparation tubes to the heating block and heat at 95° C. for 20 minutes make sure to carefully remove the tubes every 5 min and vortex each tube for 10 s and return to heating block. 
         [0092]    Remove sample preparation tube from heating block and immediately add an equivalent volume of lysis solution as the volume added of lysis solution from step 1 above. For example, if 50 μL of lysis solution was added, add 50 μL of lysis solution. 
         [0093]    Vortex each sample for 10 seconds. Spin down the sample preparation tubes in a microcentrifuge and allow to cool. Use the resultant lysis solution lysate supernatant directly in the PCR reaction. 
         [0094]    The extracted DNA needs to be diluted to a concentration of 5 ng/μl in 1×TE buffer at pH 8.0 and then stored at +4 to +8° C. for 1 week or at −20° C. if longer term storage is required. The QClamp qPCR reaction is optimized for DNA samples containing 5-20 ng of purified genomic DNA. 
         [0095]    The sequences in the Table below show exemplary primers and xeno and peptide nucleic acids. 
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 Sequence Name 
                   
                   
               
               
                   
               
             
             
               
                 1047SSF001NEW 
                 SEQ ID No: 21 
                 CGAAAGACCCTAGCCTTAGATAAAACT 
               
               
                   
               
               
                 1047SSR001NEW 
                 SEQ ID No: 22 
                 ATTGTGTGGAAGATCCAATCCATTT 
               
               
                   
               
               
                 146R002f 
                 SEQ ID No: 23 
                 ACGTTGGATGTGTACCATACCTGTCTGGTCTT 
               
               
                   
               
               
                 21FW1S 
                 SEQ ID No: 24 
                 GTTTTCCCAGTCACGACACGTTGGATGCAGCCAGGA 
               
               
                   
                   
                 ACGTACTGGTGA 
               
               
                   
               
               
                 BIOBRAFCONTRLFP 
                 SEQ ID No: 25 
                 /5Biosg/CTCCAGATCTCAGTAAGGTACGG 
               
               
                   
               
               
                 BIOKRASCONTRLFP 
                 SEQ ID No: 26 
                 /5Biosg/TGAGGGAGATCCGACAATACAG 
               
               
                   
               
               
                 BRAFAZFPNEW02 
                 SEQ ID No: 27 
                 ACAGTAAAAATAGGTGATTTTGGTCTAGCTA 
               
               
                   
               
               
                 BRAFAZFPNew02s 
                 SEQ ID No: 28 
                 GTTTTCCCAGTCACGACACGTTGGATGACAGTAAAA 
               
               
                   
                   
                 ATAGGTGATTTTGGTCTAGCTA 
               
               
                   
               
               
                 BRAFAZRP001 
                 SEQ ID No: 29 
                 CATCCACAAAATGGATCCAGACAA 
               
               
                   
               
               
                 BRAFAZRP00ls 
                 SEQ ID No: 30 
                 CAGGAAACAGCTATGACACGTTGGATGCATCCACAA 
               
               
                   
                   
                 AATGGATCCAGACAA 
               
               
                   
               
               
                 BRAFCONTRLFP 
                 SEQ ID No: 31 
                 CTCCAGATCTCAGTAAGGTACGG 
               
               
                   
               
               
                 BRAFCONTRLRP 
                 SEQ ID No: 32 
                 GGGAAAGAGTGGTCTCTCATC 
               
               
                   
               
               
                 C790F002f 
                 SEQ ID No: 33 
                 ACGTTGGATGTCCACCGTGCAGCTCATC 
               
               
                   
               
               
                 C790F002fS 
                 SEQ ID No: 34 
                 GTTTTCCCAGTCACGACACGTTGGATGTCCACCGTGC 
               
               
                   
                   
                 AGCT 
               
               
                   
               
               
                 C790R001Bf 
                 SEQ ID No: 35 
                 ACGTTGGATGGTCTTTGTGTTCCCGGACAT 
               
               
                   
               
               
                 C790R001BfS 
                 SEQ ID No: 36 
                 CAGGAAACAGCTATGACACGTTGGATGGTCTTTGTG 
               
               
                   
                   
                 TTCCC 
               
               
                   
               
               
                 Ex19NewFS 
                 SEQ ID No: 37 
                 GTTTTCCCAGTCACGACACGTTGGATGCTCTCTGTCA 
               
               
                   
                   
                 TAGGGACTCTGGATCC 
               
               
                   
               
               
                 Ex19NewFwd 
                 SEQ ID No: 38 
                 CTCTCTGTCATAGGGACTCTGGATCC 
               
               
                   
               
               
                 Ex19NewRev 
                 SEQ ID No: 39 
                 AGCAAAGCAGAAACTCACATCGAG 
               
               
                   
               
               
                 Ex19NewRS 
                 SEQ ID No: 40 
                 CAGGAAACAGCTATGACACGTTGGATGAGCAAAGCA 
               
               
                   
                   
                 GAAACTCACATCGAG 
               
               
                   
               
               
                 Exon18NewFS 
                 SEQ ID No: 41 
                 GTTTTCCCAGTCACGACACGTTGGATGGCTCCCAACC 
               
               
                   
                   
                 AAGCTCTCTTGA 
               
               
                   
               
               
                 Exon18NewFwd 
                 SEQ ID No: 42 
                 GCTCCCAACCAAGCTCTCTTGA 
               
               
                   
               
               
                 Exon18NewRev 
                 SEQ ID No: 43 
                 CTGTGCCAGGGACCTTACCTTATAC 
               
               
                   
               
               
                 Exon18NewRS 
                 SEQ ID No: 44 
                 CAGGAAACAGCTATGACACGTTGGATGCTGTGCCAG 
               
               
                   
                   
                 GGACCTTACCTTATAC 
               
               
                   
               
               
                 Exon2FowardNew 
                 SEQ ID No: 45 
                 TTTGCCAAGGCACGAGTAACAAG 
               
               
                   
               
               
                 Exon2ReverseNew 
                 SEQ ID No: 46 
                 CCCAAGGACCACCTCACAGTTAT 
               
               
                   
               
               
                 JAK2XN9F001 
                 SEQ ID No: 47 
                 TTAACTGCAGATGCACATCATTACCT 
               
               
                   
               
               
                 KRAS117F002 
                 SEQ ID No: 48 
                 GGACTCTGAAGATGTACCTATGG 
               
               
                   
               
               
                 KRAS117F002s 
                 SEQ ID No: 49 
                 GTTTTCCCAGTCACGACACGTTGGATGGGACTCTGAA 
               
               
                   
                   
                 GATGTACCTATGG 
               
               
                   
               
               
                 KRAS117R002 
                 SEQ ID No: 50 
                 GCTAAGTCCTGAGCCTGTTT 
               
               
                   
               
               
                 KRAS117R002s 
                 SEQ ID No: 51 
                 CAGGAAACAGCTATGACACGTTGGATGGCTAAGTCC 
               
               
                   
                   
                 TGAGCCTGTTT 
               
               
                   
               
               
                 KRAS146F003 
                 SEQ ID No: 52 
                 ACACAAAACAGGCTCAGGAC 
               
               
                   
               
               
                 KRAS146F003s 
                 SEQ ID No: 53 
                 GTTTTCCCAGTCACGACACGTTGGATGACACAAAAC 
               
               
                   
                   
                 AGGCTCAGGAC 
               
               
                   
               
               
                 KRAS146R002 
                 SEQ ID No: 54 
                 CAGTGTTACTTACCTGTCTTGTCTT 
               
               
                   
               
               
                 KRAS146R002s 
                 SEQ ID No: 55 
                 CAGGAAACAGCTATGACACGTTGGATGCAGTGTTAC 
               
               
                   
                   
                 TTACCTGTCTTGTCTT 
               
               
                   
               
               
                 KRASBIOFP002 
                 SEQ ID No: 56 
                 AAGGCCTGCTGAAAATGACTG 
               
               
                   
               
               
                 KRASBioFP002s 
                 SEQ ID No: 57 
                 GTTTTCCCAGTCACGACACGTTGGATGAAGGCCTGCT 
               
               
                   
                   
                 GAAAATGACTG 
               
               
                   
               
               
                 KRASC12RP002s 
                 SEQ ID No: 58 
                 CAGGAAACAGCTATGACACGTTGGATGTCAAGGCAC 
               
               
                   
                   
                 TCTTGCCTACGC 
               
               
                   
               
               
                 KRASc13F001 
                 SEQ ID No: 59 
                 ACTTGTGGTAGTTGGAGCTGGT 
               
               
                   
               
               
                 KRASC13F001s 
                 SEQ ID No: 60 
                 GTTTTCCCAGTCACGACACGTTGGATGACTTGTGGTA 
               
               
                   
                   
                 GTTGGAGCTGGT 
               
               
                   
               
               
                 KRASC13NEWR001 
                 SEQ ID No: 61 
                 TCATGAAAATGGTCAGAGAAACCTT 
               
               
                   
               
               
                 KRASC13NewR001s 
                 SEQ ID No: 62 
                 CAGGAAACAGCTATGACACGTTGGATGACTTGTGGT 
               
               
                   
                   
                 AGTTGGAGCTGGT 
               
               
                   
               
               
                 KRASC59R001 
                 SEQ ID No: 63 
                 ATTGCACTGTACTCCTCTTGACC 
               
               
                   
               
               
                 KRASC59R001s 
                 SEQ ID No: 64 
                 CAGGAAACAGCTATGACACGTTGGATGATTGCACTG 
               
               
                   
                   
                 TACTCCTCTTGACC 
               
               
                   
               
               
                 KRASc61F001 
                 SEQ ID No: 65 
                 CTCTTGGATATTCTCGACACAGCAGGT 
               
               
                   
               
               
                 KRASC61F001s 
                 SEQ ID No: 66 
                 GTTTTCCCAGTCACGACACGTTGGATGCTCTTGGATA 
               
               
                   
                   
                 TTCTCGACACAGCAGGT 
               
               
                   
               
               
                 KRASc61F003 
                 SEQ ID No: 67 
                 CCAGACTGTGTTTCTCCCTT 
               
               
                   
               
               
                 KRASC61F003s 
                 SEQ ID No: 68 
                 GTTTTCCCAGTCACGACACGTTGGATGCCAGACTGTG 
               
               
                   
                   
                 TTTCTCCCTT 
               
               
                   
               
               
                 KRASCONTRLFP 
                 SEQ ID No: 69 
                 TGAGGGAGATCCGACAATACAG 
               
               
                   
               
               
                 KRASCONTRLRP 
                 SEQ ID No: 70 
                 TCTGCCAAAATTAATGTGCTGAACT 
               
               
                   
               
               
                 L858RBR001 
                 SEQ ID No: 71 
                 TTCTCTTCCGCACCCAGC 
               
               
                   
               
               
                 L858RBR001S 
                 SEQ ID No: 72 
                 CAGGAAACAGCTATGACACGTTGGATGTTCTCTTCCG 
               
               
                   
                   
                 CACCCAGC 
               
               
                   
               
               
                 L858RNewFS 
                 SEQ ID No: 73 
                 GTTTTCCCAGTCACGACACGTTGGATGTGAAAACAC 
               
               
                   
                   
                 CGCAGCATGTCAAGA 
               
               
                   
               
               
                 L858RNewFwd 
                 SEQ ID No: 74 
                 TGAAAACACCGCAGCATGTCAAGA 
               
               
                   
               
               
                 L858RNewRev 
                 SEQ ID No: 75 
                 CCTTACTTTGCCTCCTTCTGCATG 
               
               
                   
               
               
                 L858RNewRS 
                 SEQ ID No: 76 
                 CAGGAAACAGCTATGACACGTTGGATGCCTTACTTT 
               
               
                   
                   
                 GCCTCCTTCTGCATG 
               
               
                   
               
               
                 NC12FP004 
                 SEQ ID No: 77 
                 TGGTGGGATCATATTCATCTACAAAG 
               
               
                   
               
               
                 NC12FP004_G13_Rev 
                 SEQ ID No: 78 
                 TGGTGGGATCATATTCATCTACAAAG 
               
               
                   
               
               
                 NC12FP004s 
                 SEQ ID No: 79 
                 CAGGAAACAGCTATGACACGTTGGATGTGGTGGGAT 
               
               
                   
                   
                 CATATTCATCTACAAAG 
               
               
                   
               
               
                 NRAS117F001 
                 SEQ ID No: 80 
                 AGTAAAAGACTCGGATGATGTACCTAT 
               
               
                   
               
               
                 NRAS117F002f 
                 SEQ ID No: 81 
                 ACGTTGGATGACCTATGGTGCTAGTGGGAAAC 
               
               
                   
               
               
                 NRAS117F003 
                 SEQ ID No: 82 
                 ACGTTGGATGTCCCGTTTTTAGGGAGCAGA 
               
               
                   
               
               
                 NRAS117F004 
                 SEQ ID No: 83 
                 CCCGTTTTTAGGGAGCAGAT 
               
               
                   
               
               
                 NRAS117R002 
                 SEQ ID No: 84 
                 CAGTTCGTGGGCTTGTTTTG 
               
               
                   
               
               
                 NRAS117R004 
                 SEQ ID No: 85 
                 CTTGCACAAATGCTGAAAGC 
               
               
                   
               
               
                 NRASc12F001 
                 SEQ ID No: 86 
                 AAACTGGTGGTGGTTGGAGCA 
               
               
                   
               
               
                 NRASC12F001s 
                 SEQ ID No: 87 
                 GTTTTCCCAGTCACGACACGTTGGATGAAACTGGTG 
               
               
                   
                   
                 GTGGTTGGAGCA 
               
               
                   
               
               
                 NRASC13F001 
                 SEQ ID No: 88 
                 GGTGGTGGTTGGAGCAGGT 
               
               
                   
               
               
                 NRASC13F001s 
                 SEQ ID No: 89 
                 GTTTTCCCAGTCACGACACGTTGGATGGGTGGTGGTT 
               
               
                   
                   
                 GGAGCAGGT 
               
               
                   
               
               
                 NRASC59F001 
                 SEQ ID No: 90 
                 ACACCCCCAGGATTCTTACAGA 
               
               
                   
               
               
                 NRASC59F001s 
                 SEQ ID No: 91 
                 GTTTTCCCAGTCACGACACGTTGGATGACACCCCCAG 
               
               
                   
                   
                 GATTCTTACAGA 
               
               
                   
               
               
                 NRASC59R001 
                 SEQ ID No: 92 
                 ATGGCACTGTACTCTTCTTGTCC 
               
               
                   
               
               
                 NRASC59R001s 
                 SEQ ID No: 93 
                 CAGGAAACAGCTATGACACGTTGGATGATGGCACTG 
               
               
                   
                   
                 TACTCTTCTTGTCC 
               
               
                   
               
               
                 NRASc61F001 
                 SEQ ID No: 94 
                 GTTGGACATACTGGATACAGCTGGA 
               
               
                   
               
               
                 NRASC61F001s 
                 SEQ ID No: 95 
                 GTTTTCCCAGTCACGACACGTTGGATGGTTGGACATA 
               
               
                   
                   
                 CTGGATACAGCTGGA 
               
               
                   
               
               
                 NRASXN3REVSet4 
                 SEQ ID No: 96 
                 CCGCAAATGACTTGCTATTA 
               
               
                   
               
               
                 NRASXN3RevSet4s 
                 SEQ ID No: 97 
                 CAGGAAACAGCTATGACACGTTGGATGCCGCAAATG 
               
               
                   
                   
                 ACTTGCTATTA 
               
               
                   
               
               
                 NRASXN5FwSet1 
                 SEQ ID No: 98 
                 ACACACTGGTAAGAGAAATAC 
               
               
                   
               
               
                 NRASXN5REVSet1 
                 SEQ ID No: 99 
                 CTGAGTCCCATCATCACT 
               
               
                   
               
               
                 BR001 
                 SEQ ID No: 100 
                 ATCGAGATTTCACTGTAGCTAGAC 
               
               
                   
               
               
                 DPCA001 
                 SEQ ID No: 101 
                 ACTTCAGGCAGCGTCTTCA 
               
               
                   
               
               
                 DPCA002 
                 SEQ ID No: 102 
                 TGTTCAGAGCACACTTCAG 
               
               
                   
               
               
                 DPCA003 
                 SEQ ID No: 103 
                 CTGGTGGTTGAATTTGCTG 
               
               
                   
               
               
                 DPCA004 
                 SEQ ID No: 104 
                 CATGAGCTCCAGCAGGATGAAC 
               
               
                   
               
               
                 DPCA005 
                 SEQ ID No: 105 
                 CCGAAGTCTCCAATCTTGG 
               
               
                   
               
               
                 DPCA006 
                 SEQ ID No: 106 
                 TAGATGTCTCGGGCCATCC 
               
               
                   
               
               
                 DPCBRC001 
                 SEQ ID No: 107 
                 GGGACACTCTAAGAT 
               
               
                   
               
               
                 DPCBRC002 
                 SEQ ID No: 108 
                 TTCTGTCCTGGGATTCTC 
               
               
                   
               
               
                 DPCBRC003 
                 SEQ ID No: 109 
                 AGATTTTCCACTTGCTGT 
               
               
                   
               
               
                 DPCBRCA001-2 
                 SEQ ID No: 110 
                 CCAGATGGGACACTCTAAGATTTTC 
               
               
                   
               
               
                 DPCBRCA002-2 
                 SEQ ID No: 111 
                 CCTTTCTGTCCTGGGATTCTCTT 
               
               
                   
               
               
                 DPCBRCA003-2 
                 SEQ ID No: 112 
                 GACAGATTTTCCACTTGCTGTGCTAA 
               
               
                   
               
               
                 DPCBRCA004 
                 SEQ ID No: 113 
                 CATAAAGGACACTGTGAAGGCC 
               
               
                   
               
               
                 DPCBRCA004B 
                 SEQ ID No: 114 
                 D-LYS-O-GGCCTTCACAGTGTCCTTTATG 
               
               
                   
               
               
                 DPCCKT002 
                 SEQ ID No: 115 
                 D-LYS-O-CATTCTTGATGTCTCTGGCTAG 
               
               
                   
               
               
                 DPCE001 
                 SEQ ID No: 116 
                 GAGCCCAGCACTTT 
               
               
                   
               
               
                 DPCE001B 
                 SEQ ID No: 117 
                 D-LYS-O-CGGAGCCCAGCACTTTGAT 
               
               
                   
               
               
                 DPCE001B1 
                 SEQ ID No: 118 
                 D-LYS-O-CGGAGCCCAGCACTTTGAT 
               
               
                   
               
               
                 DPCE002 
                 SEQ ID No: 119 
                 NH(2)-AGATGTTGCTTCTCTTAA-CONH(2) 
               
               
                   
               
               
                 DPCE002B 
                 SEQ ID No: 120 
                 D-LYS-O-AGATGTTGCTTCTCTTAA 
               
               
                   
               
               
                 DPCE002C 
                 SEQ ID No: 121 
                 D-LYS-O-CGGAGATGTTGCTTCTCTTAATTCC 
               
               
                   
               
               
                 DPCE004 
                 SEQ ID No: 122 
                 CAGTTTGGCCAGCCCA 
               
               
                   
               
               
                 DPCE004B 
                 SEQ ID No: 123 
                 CAGTTTGGCCAGCCCA-O-D-LYS 
               
               
                   
               
               
                 DPCE004C 
                 SEQ ID No: 124 
                 D-LYS-O-TTTGGCCAGCCCAAAATCTGT 
               
               
                   
               
               
                 DPCE004D 
                 SEQ ID No: 125 
                 D-LYS-O-GGCCAGCCCAAAATCTGT 
               
               
                   
               
               
                 DPCE005 
                 SEQ ID No: 126 
                 ACCCAGCAGTTTGGC 
               
               
                   
               
               
                 DPCE005B 
                 SEQ ID No: 127 
                 D-LYS-O-ACCCAGCAGTTTGGC 
               
               
                   
               
               
                 DPCE006 
                 SEQ ID No: 128 
                 GCTGCGTGATGAG 
               
               
                   
               
               
                 DPCE007 
                 SEQ ID No: 129 
                 GCTGCGTGATGA 
               
               
                   
               
               
                 DPCE008 
                 SEQ ID No: 130 
                 AGCTCATCACGCAGCTCATG 
               
               
                   
               
               
                 DPCE008B 
                 SEQ ID No: 131 
                 D-LYS-O-CAGCTCATCACGCAGCTCATGC 
               
               
                   
               
               
                 DPCE008C 
                 SEQ ID No: 132 
                 D-LYS-O-TCATCACGCAGCTCATGCCCTT 
               
               
                   
               
               
                 DPCE008D 
                 SEQ ID No: 133 
                 D-LYS-O-CTCATCACGCAGCTCATG 
               
               
                   
               
               
                 DPCE008E 
                 SEQ ID No: 134 
                 D-LYS-O-TGAGCTGCGTGATG 
               
               
                   
               
               
                 DPCE009B 
                 SEQ ID No: 135 
                 D-LYS-O-TCCACGCTGGCCATCACGTA 
               
               
                   
               
               
                 DPCE009B-1 
                 SEQ ID No: 136 
                 TCCACGCTGGCCATCACGTA-O-D-LYS 
               
               
                   
               
               
                 DPCE010B 
                 SEQ ID No: 137 
                 TGGGGGTTGTCCAC-O-D-LYS 
               
               
                   
               
               
                 DPCE011 
                 SEQ ID No: 138 
                 GCACACGTGGGGGTT-O-D-LYS 
               
               
                   
               
               
                 DPCE012 
                 SEQ ID No: 139 
                 D-LYS-O-ACAACCCCCACGTGTGC 
               
               
                   
               
               
                 DPCH001 
                 SEQ ID No: 140 
                 CTGAGCCAGGAGAAAC 
               
               
                   
               
               
                 DPCH002 
                 SEQ ID No: 141 
                 GTAAACTGAGCCAGGAG 
               
               
                   
               
               
                 DPCH003 
                 SEQ ID No: 142 
                 ATGGCACTAGTAAACTGAGC 
               
               
                   
               
               
                 DPCH004 
                 SEQ ID No: 143 
                 ATCCATATAACTGAAAGCCAA 
               
               
                   
               
               
                 DPCH005 
                 SEQ ID No: 144 
                 ACCACATCATCCATATAACTGAA 
               
               
                   
               
               
                 DPCHRAS001B 
                 SEQ ID No: 145 
                 D-LYS-O-O-TTGCCCACACCGCCGGC 
               
               
                   
               
               
                 DPCHRAS002 
                 SEQ ID No: 146 
                 D-LYS-O-O-TCTTGCCCACACCGCC 
               
               
                   
               
               
                 DPCHRAS003 
                 SEQ ID No: 147 
                 D-LYS-O-O-TACTCCTCCTGGCCGGC 
               
               
                   
               
               
                 DPCJ001 
                 SEQ ID No: 148 
                 CGTCTCCACAGACACATACTCCA 
               
               
                   
               
               
                 DPCJ002B 
                 SEQ ID No: 149 
                 CGTCTCCACAGACACATACTCCA-O-D-LYS 
               
               
                   
               
               
                 DPCK001B 
                 SEQ ID No: 150 
                 GCCTACGCCACCAGCTCCAAC-O-D-LYS 
               
               
                   
               
               
                 DPCK001B2 
                 SEQ ID No: 151 
                 GCCTACGCCACCAGCTCCAAC-O-O-D-LYS 
               
               
                   
               
               
                 DPCK001C 
                 SEQ ID No: 152 
                 CTACGCCACCAGCTCCAACTACCA 
               
               
                   
               
               
                 DPCK001C2 
                 SEQ ID No: 153 
                 CTACGCCACCAGCTCCAACTACCA-O-D-LYS 
               
               
                   
               
               
                 DPCK002 
                 SEQ ID No: 154 
                 TCTTGCCTACGCCACCAGCTCCA 
               
               
                   
               
               
                 DPCK003 
                 SEQ ID No: 155 
                 TGTACTCCTCTTGACCTGCTGTG 
               
               
                   
               
               
                 DPCK003B 
                 SEQ ID No: 156 
                 D-LYS-O-TGTACTCCTCTTGACCTGCTGTG 
               
               
                   
               
               
                 DPCK004 
                 SEQ ID No: 157 
                 NH(2)-GGCAAATCACATTTATTTCCTAC-CONH(2) 
               
               
                   
               
               
                 DPCK004B 
                 SEQ ID No: 158 
                 D-LYS-O-GGCAAATCACATTTATTTCCTAC 
               
               
                   
               
               
                 DPCK005B 
                 SEQ ID No: 159 
                 D-LYS-O-TGTCTTGTCTTTGCTGATGTTTC 
               
               
                   
               
               
                 DPCK005 
                 SEQ ID No: 160 
                 TGTCTTGTCTTTGCTGATGTTTC 
               
               
                   
               
               
                 DPCK005C 
                 SEQ ID No: 161 
                 D-LYS-O-TGTCTTGTCTTTGCTGATGTTTC 
               
               
                   
               
               
                 DPCK006 
                 SEQ ID No: 162 
                 NH(2)-CTCTTGACCTGCTGTGTCGAG-CONH(2) 
               
               
                   
               
               
                 DPCN001 
                 SEQ ID No: 163 
                 TCCCAACACCACCTGCTCCAA 
               
               
                   
               
               
                 DPCN001B 
                 SEQ ID No: 164 
                 D-LYS-O-CAACACCACCTGCTCCAACCACCAC 
               
               
                   
               
               
                 DPCN002 
                 SEQ ID No: 165 
                 CTTTTCCCAACACCACCTGCTCC 
               
               
                   
               
               
                 DPCN002B 
                 SEQ ID No: 166 
                 D-LYS-O-TGCGCTTTTCCCAACACCACCTGCT 
               
               
                   
               
               
                 DPCN003B 
                 SEQ ID No: 167 
                 GGCACTGTACTCTTCTTGTCCAG 
               
               
                   
               
               
                 DPCN004B 
                 SEQ ID No: 168 
                 D-LYS-O-TCTGGTCTTGGCTGAGGTTTC 
               
               
                   
               
               
                 DPCN006 
                 SEQ ID No: 169 
                 NH(2)-GGCAAATCACACTTGTTTCCCAC-CONH(2) 
               
               
                   
               
               
                 DPCN006B 
                 SEQ ID No: 170 
                 D-LYS-O-GGCAAATCACACTTGTTTCCCAC 
               
               
                   
               
               
                 DPCN007 
                 SEQ ID No: 171 
                 NH(2)-TTCTTGTCCAGCTGTATCCAGTATG-CONH(2) 
               
               
                   
               
               
                 DPCPKA003B 
                 SEQ ID No: 172 
                 D-LYS-O-AGATCCTCTCTCTGAAATCAC 
               
               
                   
               
               
                 DPCPKA004 
                 SEQ ID No: 173 
                 D-LYS-O-TCTTTCTCCTGCTCAGTGATTTCA 
               
               
                   
               
               
                 DPCPKA005 
                 SEQ ID No: 174 
                 D-LYS-O-AATGATGCACATCATGGTGGCTG 
               
               
                   
               
               
                 NRASN003C 
                 SEQ ID No: 175 
                 D-LYS-O-GGCACTGTACTCTTCTTGTCCAG 
               
               
                   
               
               
                 QMDXNA001 
                 SEQ ID No: 176 
                 NH(2)-O-TTCATCAACCGCACTCTGTTTATCTC 
               
               
                   
               
               
                 QMDXNA002 
                 SEQ ID No: 177 
                 NH(2)-O-TGGCGACGACAATGGACCCAATTAT 
               
               
                   
               
               
                 QMDXNA003 
                 SEQ ID No: 178 
                 NH(2)-O-AGATGTAGTTAGCAATCGGTCCTTGTTGTA 
               
               
                   
               
               
                 QMDXNA004 
                 SEQ ID No: 179 
                 NH(2)-O-GGGTAATTGAGGTAACGTAGGTATCAAGAT 
               
               
                   
               
               
                 QMDXNA005 
                 SEQ ID No: 180 
                 NH(2)-O-TACTATCGACTGACATGAGGCTTGTGT 
               
               
                   
               
               
                 XNADE001 
                 SEQ ID No: 181 
                 D-LYS-O-AGTCCGACGATCTGGAATTC 
               
               
                   
               
               
                 XNADE002 
                 SEQ ID No: 182 
                 D-LYS-O-ACTGGAGTTCAGACGTGTG 
               
               
                   
               
               
                 XNADE003 
                 SEQ ID No: 183 
                 D-LYS-O-CTCTTCCGATCAGATCGGAA 
               
               
                   
               
               
                 XNADE003b 
                 SEQ ID No: 184 
                 D-LYS-O-CTCTTCCGATCAGATCGGAAG 
               
               
                   
               
               
                 XNAFGFR001 
                 SEQ ID No: 185 
                 D-LYS-O-O-AGCGCTCCCCGCACC 
               
               
                   
               
               
                 XNAFGFR001 
                 SEQ ID No: 186 
                 D-LYS-O-O-AGCGCTCCCCGCACC 
               
               
                   
               
               
                 XNAFGFR002 
                 SEQ ID No: 187 
                 D-LYS-O-GGGGAGCGCTCTGT-O-TTTTT 
               
               
                   
               
               
                 XNAFGFR003 
                 SEQ ID No: 188 
                 D-LYS-O-O-AGCGCTCCCCGCACC-O-TTTTTT 
               
               
                   
               
               
                 XNAFGFR004 
                 SEQ ID No: 189 
                 D-LYS-O-TGCATACACACTGCCCGCCT 
               
               
                   
               
             
          
         
       
     
         [0096]    Other sequences of interest in connection with the invention include the following exons: 
         [0000]    
       
         
               
               
             
           
               
                 BRAF Ex 15 NCBI NG_007873.3; DNA; Wildtype 
                   
               
               
                 SEQ ID No. 190 
                   
               
               
                 TAGAAATTAG ATCTCTTACC TAAACTCTTC ATAATGCTTG CTCTGATAGG AAAATGAGAT 
                   
               
               
                   
               
               
                 CTACTGTTTT CCTTTACTTA CTACACCTCA GATATATTTC TTCATGAAGA CCTCACAGTA 
               
               
                   
               
               
                 AAAATAGGTG ATTTTGGTCT AGCTACAGTG AAATCTCGAT GGAGTGGGTC CCATCAGTTT 
               
               
                   
               
               
                 GAACAGTTGT CTGGATCCAT TTTGTGGATG GTAAGAATTG AGGCTATTTT TCCACTGATT 
               
               
                   
               
               
                 AAATTTTTGG CCCTGAGATG CTGCTGAGTT ACTAGAAAGT CATTGAAGGT CTCAACTATA 
               
               
                   
               
               
                 GTATTTTCAT AGTTCCCAGT ATTCACAAAA ATCAGTGTTC TTATTTTTTA TGTAAATAGA 
               
               
                   
               
               
                 EGFR Ex18 GeneGenBank: AF288738.1 NCBI NM_005228.3; 
               
               
                 DNA; Wildtype 
               
               
                 SEQ ID No. 191 
                   
               
               
                 TAGAGAAGGC GTACATTTGT CCTTCCAAAT GAGCTGGCAA GTGCCGTGTC CTGGCACCCA 
                   
               
               
                   
               
               
                 AGCCCATGCC GTGGCTGCTG GTCCCCCTGC TGGGCCATGT CTGGCACTGC TTTCCAGCAT 
               
               
                   
               
               
                 GGTGAGGGCT GAGGTGACCC TTGTCTCTGT GTTCTTGTCC CCCCCAGCTT GTGGAGCCTC 
               
               
                   
               
               
                 TTACACCCAG TGGAGAAGCT CCCAACCAAG CTCTCTTGAG GATCTTGAAG GAAACTGAAT 
               
               
                   
               
               
                 TCAAAAAGAT CAAAGTGCTG GGCTCCGGTG CGTTCGGCAC GGTGTATAAG GTAAGGTCCC 
               
               
                   
               
               
                 TGGCACAGGC CTCTGGGCTG GGCCGCAGGG CCTCTCATGG TCTGGTGGGG AGCCCAGAGT 
               
               
                   
               
               
                 CCTTGCAAGC TGTATATTTC CATCATCTAC TTTACTCTTT GTTTCACTGA GTGTTTGGGA 
               
               
                   
               
               
                 AACTCCAGTG TTTTTCCCAA GTTATTGAGA GGAAATCTTT TATAACCACA GTAATCAGTG 
               
               
                   
               
               
                 EGFR Ex19 GeneGenBank: AF288738.1 NCBI NM_005228.3; 
               
               
                 DNA; Wildtype 
               
               
                 SEQ ID No. 192 
                   
               
               
                 AGCCCAACAG CTGCAGGGCT GCGGGGGCGT CACAGCCCCC AGCAATATCA GCCTTAGGTG 
                   
               
               
                   
               
               
                 CGGCTCCACA GCCCCAGTGT CCCTCACCTT CGGGGTGCAT CGCTGGTAAC ATCCACCCAG 
               
               
                   
               
               
                 ATCACTGGGC AGCATGTGGC ACCATCTCAC AATTGCCAGT TAACGTCTTC CTTCTCTCTC 
               
               
                   
               
               
                 TGTCATAGGG ACTCTGGATC CCAGAAGGTG AGAAAGTTAA AATTCCCGTC GCTATCAAGG 
               
               
                   
               
               
                 AATTAAGAGA AGCAACATCT CCGAAAGCCA ACAAGGAAAT CCTCGATGTG AGTTTCTGCT 
               
               
                   
               
               
                 TTGCTGTGTG GGGGTCCATG GCTCTGAACC TCAGGCCCAC CTTTTCTCAT GTCTGGCAGC 
               
               
                   
               
               
                 TGCTCTGCTC TAGACCCTGC TCATCTCCAC ATCCTAAATG TTCACTTTCT ATGTCTTTCC 
               
               
                   
               
               
                 EGFR Ex20 GeneGenBank: AF288738.1 NCBI NM_005228.3; 
               
               
                 DNA; Wildtype 
               
               
                 SEQ ID No. 193 
                   
               
               
                 AAAATTCCCG TCGCTATCAA GGAATTAAGA GAAGCAACAT CTCCGAAAGC CAACAAGGAA 
                   
               
               
                   
               
               
                 ATCCTCGATG AAGCCTACGT GATGGCCAGC GTGGACAACC CCCACGTGTG CCGCCTGCTG 
               
               
                   
               
               
                 GGCATCTGCC TCACCTCCAC CGTGCAGCTCATCACGCAGC TCATGCCCTT CGGCTGCCTC 
               
               
                   
               
               
                 CTGGACTATG TCCGGGAACA CAAAGACAAT ATTGGCTCCC AGTACCTGCT CAACTGGTGT 
               
               
                   
               
               
                 GTGCAGATCG CAAAGGGCAT GAACTACTTG GAGGACCGTC GCTTGGTGCA CCGCGACCTG 
               
               
                   
               
               
                 GCAGCCAGGA ACGTACTGGT GAAAACACCG CAGCATGTCA AGATCACAGA TTTTGGGCTG 
               
               
                   
               
               
                 GCCAAACTGC TGGGTGCGGA AGAGAAAGAA TACCATGCAG AAGGAGGCAA AGTGCCTATC 
               
               
                   
               
               
                 AAGTGGATGG CATTGGAATC AATTTTACAC AGAATCTATA CCCACCAGAG TGATGTCTGG 
               
               
                   
               
               
                 EGFR Ex21 GeneGenBank: AF288738.1 NCBI NM_005228.3; 
               
               
                 DNA; Wildtyp 
               
               
                 SEQ ID No. 194 
                   
               
               
                 GGCATCTGCC TCACCTCCAC CGTGCAGCTC ATCACGCAGC TCATGCCCTT CGGCTGCCTC 
                   
               
               
                   
               
               
                 CTGGACTATG TCCGGGAACA CAAAGACAAT ATTGGCTCCC AGTACCTGCT CAACTGGTGT 
               
               
                   
               
               
                 GTGCAGATCG CAAAGGGCAT GAACTACTTG GAGGACCGTC GCTTGGTGCA CCGCGACCTG 
               
               
                   
               
               
                 GCAGCCAGGA ACGTACTGGT GAAAACACCG CAGCATGTCA AGATCACAGA TTTTGGGCTG 
               
               
                   
               
               
                 GCCAAACTGC TGGGTGCGGA AGAGAAAGAA TACCATGCAG AAGGAGGCAA AGTGCCTATC 
               
               
                   
               
               
                 AAGTGGATGG CATTGGAATC AATTTTACAC AGAATCTATA CCCACCAGAG TGATGTCTGG 
               
               
                   
               
               
                 AGCTACGGGG TGACCGTTTG GGAGTTGATG ACCTTTGGAT CCAAGCCATA TGACGGAATC 
               
               
                   
               
               
                 HRAS Ex3 NCBI Reference Sequence: NG_007666.1; 
               
               
                 DNA; Wildtype 
               
               
                 SEQ ID No. 195 
                   
               
               
                 CTGCAGGATT CCTACCGGAA GCAGGTGGTC ATTGATGGGG AGACGTGCCT GTTGGACATC 
                   
               
               
                   
               
               
                 CTGGATACCG CCGGCCAGGA GGAGTACAGC GCCATGCGGG ACCAGTACAT GCGCACCGGG 
               
               
                   
               
               
                 GAGGGCTTCC TGTGTGTGTT TGCCATCAAC AACACCAAGT CTTTTGAGGA CATCCACCAG 
               
               
                   
               
               
                 TACAGGTGAA CCCCGTGAGG CTGGCCCGGG AGCCCACGCC GCACAGGTGG GGCCAGGCC 
               
               
                   
               
               
                 JAK2 NCBI Reference Sequence: NG_009904.1; 
               
               
                 DNA; wildtype 
               
               
                 SEQ ID No. 196 
                   
               
               
                 CTGACATCTACCTCTAGTTGTACTTCTGTCCTCTATTTCAGGTGTTATGGGTCAAGCCTGTTTGACTGGC 
                   
               
               
                   
               
               
                 ATTATTCATGATTCCTGTACCACTCTTGCTCTCTCTCACTTTGATCTCCATATTCCAGGCTTACACAGGG 
               
               
                   
               
               
                 GTTTCCTCAGAACGTTGATGGCAGTTGCAGGTCCATATAAAGGGACCAAAGCACATTGTATCCTCATC 
               
               
                   
               
               
                 TAGTCATGCTGAAAGTAGGAGAAAGTGCATCTTTATTATGGCAGAGAGAATTTTCTGAACTATTTATG 
               
               
                   
               
               
                 GACAACAGTCAAACAACAATTCTTTGTACTTTTTTTTTTCCTTAGTCTTTCTTTGAAGCAGCAAGTATGA 
               
               
                   
               
               
                 TGAGCAAGCTTTCTCACAAGCATTTGGTTTTAAATTATGGAGTATGTGTCTGTGGAGACGAGAGTAAG 
               
               
                   
               
               
                 TAAAACTACAGGCTTTCTAATGCCTTTCTCAGAGCATCTGTTTTTGTTTATATAGAAAATTCAGTTTCAG 
               
               
                   
               
               
                 GATCACAGCTAGGTGTCAGTGTAAACTATAATTTAACAGGAGTTAAGTATTTTTGAAACTGAAAACAC 
               
               
                   
               
               
                 TGTAGGACTATTCAGTTATATCTTGTGAAAAAGGAAAGCAATGAAGTTAAAAGTAGAAGGTTACAATG 
               
               
                   
               
               
                 CCCAAACAATAGAGTATTATAGTAAACAAATGTCTATAAAACATTTTGTGTTCATGATAGCAAAAGAG 
               
               
                   
               
               
                 ATTATGGCAGGTTCAACATAACATTGGAATAACTGGCCTTTTCAGTACAAACTTATCTGGAATTATGAA 
               
               
                   
               
               
                 GACAAAGCATA 
               
               
                   
               
               
                 KRAS Ex2 NCBI Reference Sequence: NG_007524.1; 
               
               
                 DNA; wildtype 
               
               
                 SEQ ID No. 197 
                   
               
               
                 GGTACTGGTG GAGTATTTGA TAGTGTATTA ACCTTATGTG TGACATGTTC TAATATAGTC 
                   
               
               
                   
               
               
                 ACATTTTCAT TATTTTTATT ATAAGGCCTG CTGAAAATGA CTGAATATAA ACTTGTGGTA 
               
               
                   
               
               
                 GTTGGAGCTG GTGGCGTAGG CAAGAGTGCC TTGACGATAC AGCTAATTCA GAATCATTTT 
               
               
                   
               
               
                 GTGGACGAAT ATGATCCAAC AATAGAGGTA AATCTTGTTT TAATATGCAT ATTACTGGTG 
               
               
                   
               
               
                 KRAS Ex3 NCBI Reference Sequence: NG_007524.1; 
               
               
                 DNA; wildtype 
               
               
                 SEQ ID No. 198 
                   
               
               
                 CTTCTCAGGA TTCCTACAGG AAGCAAGTAG TAATTGATGG AGAAACCTGT CTCTTGGATA 
                   
               
               
                   
               
               
                 TTCTCGACAC AGCAGGTCAA GAGGAGTACA GTGCAATGAG GGACCAGTAC ATGAGGACTG 
               
               
                   
               
               
                 GGGAGGGCTT TCTTTGTGTA TTTGCCATAA ATAATACTAA ATCATTTGAA GATATTCACC 
               
               
                   
               
               
                 ATTATAGGTG GGTTTAAATT GAATATAATA AGCTGACATT AAGGAGTAAT TATAGTTTTT 
               
               
                   
               
               
                 KRAS Ex4 NCBI Reference Sequence: NG_007524.1; 
               
               
                 DNA; wildtype 
               
               
                 SEQ ID No. 199 
                   
               
               
                 GTGCTATAAC TTTTTTTTCT TTCCCAGAGA ACAAATTAAA AGAGTTAAGG ACTCTGAAGA 
                   
               
               
                   
               
               
                 TGTACCTATG GTCCTAGTAG GAAATAAATG TGATTTGCCT TCTAGAACAG TAGACACAAA 
               
               
                   
               
               
                 ACAGGCTCAG GACTTAGCAA GAAGTTATGG AATTCCTTTT ATTGAAACAT CAGCAAAGAC 
               
               
                   
               
               
                 AAGACAGGTA AGTAACACTG AAATAAATAC AGATCTGTTT TCTGCAAAAT CATAACTGTT 
               
               
                   
               
               
                 ATGTCATTTA ATATATCAGT TTTTCTCTCA ATTATGCTAT ACTAGGAAAT AAAACAATAT 
               
               
                   
               
               
                 KRAS Ex5 NCBI Reference Sequence: NG_007524.1; 
               
               
                 DNA; wildtype 
               
               
                 SEQ ID No. 200 
                   
               
               
                 AATGCAACAG ACTTTAAAGA AGTTGTGTTT TACAATGCAG AGAGTGGAGG ATGCTTTTTA 
                   
               
               
                   
               
               
                 TACATTGGTG AGGGAGATCC GACAATACAG ATTGAAAAAA ATCAGCAAAG AAGAAAAGAC 
               
               
                   
               
               
                 TCCTGGCTGT GTGAAAATTA AAAAATGCAT TATAATGTAA TCTGGTAAGT TTAAGTTCAG 
               
               
                   
               
               
                 NRAS Exon 2 NCBI Reference Sequence: NG_007572.1; 
               
               
                 DNA; wildtype 
               
               
                 SEQ ID No. 201 
                   
               
               
                 GTGTTTTTGC GTTCTCTAGT CACTTTAAGA ACCAAATGGA AGGTCACACT AGGGTTTTCA 
                   
               
               
                   
               
               
                 TTTCCATTGA TTATAGAAAG CTTTAAAGTA CTGTAGATGT GGCTCGCCAA TTAACCCTGA 
               
               
                   
               
               
                 TTACTGGTTT CCAACAGGTT CTTGCTGGTG TGAAATGACT GAGTACAAAC TGGTGGTGGT 
               
               
                   
               
               
                 TGGAGCAGGT GGTGTTGGGA AAAGCGCACT GACAATCCAG CTAATCCAGA ACCACTTTGT 
               
               
                   
               
               
                 AGATGAATAT GATCCCACCA TAGAGGTGAG GCCCAGTGGT AGCCCGCTGA CCTGATCCTG 
               
               
                   
               
               
                 TCTCTCACTT GTCGGATCAT CTTTACCCAT ATTCTGTATT AAAGGAATAA GAGGAGAGAA 
               
               
                   
               
               
                 AGTAAAAAGT TATTTTGGGT ATACATTCAG TTATGCAATA AGCTTAACGT GTTTATAGAG 
               
               
                   
               
               
                 AACAGTTCAT TTTTATTAGC TGCTGAAGTT TCTAAAACCT GTCCAGTTTT TAACAGTTCT 
               
               
                   
               
               
                 NRAS Exon 3 NCBI Reference Sequence: NG_007572.1; 
               
               
                 DNA; wildtype 
               
               
                 SEQ ID No. 202 
                   
               
               
                 TGGGCTTGAA TAGTTAGATG CTTATTTAAC CTTGGCAATA GCATTGCATT CCCTGTGGTT 
                   
               
               
                   
               
               
                 TTTAATAAAA ATTGAACTTC CCTCCCTCCC TGCCCCCTTA CCCTCCACAC CCCCAGGATT 
               
               
                   
               
               
                 CTTACAGAAA ACAAGTGGTT ATAGATGGTG AAACCTGTTT GTTGGACATA CTGGATACAG 
               
               
                   
               
               
                 CTGGACAAGA AGAGTACAGT GCCATGAGAG ACCAATACAT GAGGACAGGC GAAGGCTTCC 
               
               
                   
               
               
                 TCTGTGTATT TGCCATCAAT AATAGCAAGT CATTTGCGGA TATTAACCTC TACAGGTACT 
               
               
                   
               
               
                 AGGAGCATTA TTTTCTCTGA AAGGATGATC TTTGTGTTCT GAATCTTTAT GGGGAAATGA 
               
               
                   
               
               
                 GGTTACCACA CTAGGGAAGA TAGAGCTTTT TAATTATGGG AAGAGTTGGT TTTAGGTTGT 
               
               
                   
               
               
                 TTGACATTGA GAATCTAGGG TAATTACTGA AAGTTAATAC TGGAATTTAT TTTACATAAT 
               
               
                   
               
               
                 NRAS Exon 4 NCBI Reference Sequence: NG_007572.1; 
               
               
                 DNA; wildtype 
               
               
                 SEQ ID No. 203 
                   
               
               
                 TGGATACAGC TGGACAAGAA GAGTACAGTG CCATGAGAGA CCAATACATG AGGACAGGCG 
                   
               
               
                   
               
               
                 AAGGCTTCCT CTGTGTATTT GCCATCAATA ATAGCAAGTC ATTTGCGGAT ATTAACCTCT 
               
               
                   
               
               
                 ACAGGGAGCA GATTAAGCGA GTAAAAGACT CGGATGATGT ACCTATGGTG CTAGTGGGAA 
               
               
                   
               
               
                 ACAAGTGTGA TTTGCCAACA AGGACAGTTG ATACAAAACA AGCCCACGAA CTGGCCAAGA 
               
               
                   
               
               
                 GTTACGGGAT TCCATTCATT GAAACCTCAG CCAAGACCAG ACAGGGTGTT GAAGATGCTT 
               
               
                   
               
               
                 TTTACACACTGGTAAGAGAA ATACGCCAGT ACCGAATGAA AAAACTCAAC AGCAGTGATG 
               
               
                   
               
               
                 ATGGGACTCA GGGTTGTATG GGATTGCCAT GTGTGGTGAT GTAACAAGAT ACTTTTAAAG 
               
               
                   
               
               
                 PIK3CA Ex9 NCBI Reference Sequence: NG_012113.2; 
               
               
                 DNA; wildtype 
               
               
                 SEQ ID No. 204 
                   
               
               
                 TGTAAAATTT ATTGAAAATG TATTTGCTTT TTCTGTAAAT CATCTGTGAA TCCAGAGGGG 
                   
               
               
                   
               
               
                 AAAAATATGA CAAAGAAAGC TATATAAGAT ATTATTTTAT TTTACAGAGT AACAGACTAG 
               
               
                   
               
               
                 CTAGAGACAA TGAATTAAGG GAAAATGACA AAGAACAGCT CAAAGCAATT TCTACACGAG 
               
               
                   
               
               
                 ATCCTCTCTC TGAAATCACT GAGCAGGAGA AAGATTTTCT ATGGAGTCAC AGGTAAGTGC 
               
               
                   
               
               
                 TAAAATGGAG ATTCTCTGTT TCTTTTTCTT TATTACAGAA AAAATAACTG AATTTGGCTG 
               
               
                   
               
               
                 ATCTCAGCAT GTTTTTACCA TACCTATTGG AATAAATAAA GCAGAATTTA CATGATTTTT 
               
               
                   
               
               
                 PIK3CA Ex20NCBI Reference Sequence: NG_012113.2; 
               
               
                 DNA; wildtype 
               
               
                 SEQ ID No. 205 
                   
               
               
                 TAGCTATTCG ACAGCATGCC AATCTCTTCA TAAATCTTTT CTCAATGATG CTTGGCTCTG 
                   
               
               
                   
               
               
                 GAATGCCAGA ACTACAATCT TTTGATGACA TTGCATACAT TCGAAAGACC CTAGCCTTAG 
               
               
                   
               
               
                 ATAAAACTGA GCAAGAGGCT TTGGAGTATT TCATGAAACA AATGAATGAT GCACATCATG 
               
               
                   
               
               
                 GTGGCTGGAC AACAAAAATG GATTGGATCT TCCACACAAT TAAACAGCAT GCATTGAACT 
               
               
                   
               
               
                 GAAAAGATAA CTGAGAAAAT GAAAGCTCAC TCTGGATTCC ACACTGCACT GTTAATAACT 
               
               
                   
               
               
                 PIK3CA Ex16 NCBI Reference Sequence: NG_012113.2; 
               
               
                 DNA; wildtype 
               
               
                 SEQ ID No. 206 
                   
               
               
                 GTTGTAAATCTTTGTAACACTTCAAAAAGCTATATTGTATTTATATTTTAAAATAAATTTCAGGGTAAA 
                   
               
               
                   
               
               
                 ATAATAATAAAGCAAAGGTACCTAGTAAAGTTTTTAACTATTTTAAAGGCTTGAAGAGTGTCGAATTA 
               
               
                   
               
               
                 TGTCCTCTGCAAAAAGGCCACTGTGGTTGAATTGGGAGAACCCAGACATCATGTCAGAGTTACTGTTT 
               
               
                   
               
               
                 CAGAACAATGAGATCATCTTTAAAAATGGGGATGGTAAGGAAGAGTATTAATGAGCTTATGATGCATG 
               
               
                   
               
               
                 AATTTAGCTATCTTTTTATACACAGGATATTTATGAACCATGAAAACTACTGAAAGCCATTTAAGGAAT 
               
               
                   
               
               
                 ATACACATGTGATAAAATATGTAATATTTATCAGATGTCTTGACCTTTGAAATATGCATGTATAATCAA 
               
               
                   
               
               
                 TGAAAAGAAAAGAAGTACTAGGTTTAGATCAGAAGTCCTGAAATCAGTTTTTTGTTTTTTCTTTTTCCT 
               
               
                   
               
               
                 GTTCCCTGCC 
               
             
          
         
       
     
         [0097]    All literature and similar materials cited in this application including, but not limited to, patents, patent applications, articles, books, treatises, and internet web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose as if they were entirely denoted. In the event that one or more of the incorporated literature and similar materials defines or uses a term in such a way that it contradicts that term&#39;s definition in this application, this application controls. 
         [0098]    Although the foregoing description contains many specifics, these should not be construed as limiting the scope of the present invention, but merely as providing illustrations of some of the presently preferred embodiments. Similarly, other embodiments may be devised without departing from the spirit or scope of the present invention. Features from different embodiments may be employed in combination. The scope of the invention is, therefore, indicated and limited only by the appended claims and their legal equivalents rather than by the foregoing description. All additions, deletions and modifications to the invention as disclosed herein which fall within the meaning and scope of the claims are to be embraced thereby.