AU2014100267A4 - Multiplexed KRAS mutation detection assay - Google Patents

Abstract

Provided herein is reagent mixture comprising multiplexed amplification reagents and flap assay reagents for detecting, in a single reaction, mutant copies of the KRAS gene that contain any of the 34A, 34C, 34T, 35A, 35C, 35T and 38A point mutations. Methods that employ the reagent mix and kits for performing the same are also provided. S38 36 or32 mutant x >J wild type x 342

Description

MULTIPLEXED KRAS MUTATION DETECTION ASSAY CROSS REFERENCE TO RELATED APPLICATIONS 5 Pursuant to 35 US.C, § 119(e), this application claims priority to the filing date of United States Provisional Patent Application Serial No, 61/548,639 filed October 18, 201 1; the disclosure of which application is herein. incorporated by reference. BACKGROUND 10 Germline KRAS mutations have been found to be associated with Noonan syndrome (Schubbert et al, Nat, Genet 2006 38 331-6) and cardio-facio-cutaneous syndrome (Niihori et al Nat, Genet. 20X6'18:294-6). Likewise, somatic KRAS mutations are found at high rates in leukemias, colorectal cancer (Bumier et al. Proc. Nat]. Acad. Sci, 1989 86: 2403-7), pancreatic cancer (Almoguera et al Cell 1988 53: 549-54) and lung cancer (Tam et al. Clin. 15 Cancer Res, 2006 12 1647-53). Methods for the detection of point mutations in KRAS may be used, for example, to provide a diagnostic for cancer and other diseases. SUMMARY Provided herein is reagent mixture comprising multiplexed amplification reagents and 20 flap assay reagents for detecting, in a single reaction, mutant copies of the KRAS gene that contain any of the 34A, 34C, 34T. 35A, 35C, 35T or 38A point mutations. Methods that employ the reagent mix and kits for performing the same are also provided, BRIEF DESCRIPTION OF THE FIGURES Fig. I schematically illustrates some of the general principles of a flap assay. Fig. 2 schematically illustrates some of the general principles or one aspect of the subject method. 30 Fig. 3 schematically illustrates some of the general principles of an example of a subject assay.

Fig 4 shows standard curves for both KRAS mutation calibrators, 35C reporting to HEX (Yellow; bottom line)) and 38A reporting to FAM (Green; top line)), and the ACTB calibrator reporting to Quasar 670 (Red; middle line), show good linearity across 5-logs, from 100000 copies per reaction to 10 copies per reaction. All three markers show similar slopes 5 and intercept values. Fig. 5 is a graph showing the distribution of percent mutation by sample type. Fig. 6 shows tables 3-6. to Fig. 7 shows the oligonucleotides used for multiplex detection and quantification of the seven mutant alel es of KRAS and the ACTB beta acting) internal control. From top to bottom, SEQ ID NO: 30, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 4(left) SEQ ID NO: 8 (rig SEQ I NC): 23, SEQ ID NC): 15 SEQ ID NO: 5 (left). SEQ I) NO: 8 (right), 15 SEQ ID NO: 16, SEQ ID NO' 24. SEQ ID NO: 6 (left), SEQ ID NO 8 (right, SEQ ID NO: 13, SEiQ lD NO: SEQ ID NO: I (left), SEQ ID NO (right), SEQ ID NO' II, SEQ ID NU; 26 SEQ ID NO: 2 (left), SEQ ID NO: 8 (right, SEQ ID NO: 12, SEQ ID NO; 27, SEQ ID NO: 3 (left), SEQ ID NO; 8 (right), SEQ ID NO; 14, SEQ ID NO: 28, SEQ ID NC:7 (eft). SEQ ID NO: 8 (right), SEQ ID NO: 18, SEQ ID NO: 29, SEQ ID NO: 9 (left), SEQ ID 20 NO: l0 (right), SEQ ID NO: 19, SEQ ID) NO: 20, and SEQ IT) NO: 2L DEFINITIONS The term "sample" as used herein relates to a material or mixture of materials, 25 typically, although not necessarily, in liquid form, containing one0 or more anaitlyes of interest. The term "nucleotide" is intended to include those moieties that contain not only the known purine and pyrimidine bases, hut also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrinidines, acylated purmes or 3(0 pyrimidines, alkylatd riboses or other beterocycles. In addition, the term "nucleotide" includes those moieties that contain hapten or fluorescent labels and may contain not only con ventional ribose and deoxyribose sugars, but other suars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety e-g. wherein one or more of 2 the hydroxyl groups are replaced with halogen atoms or aliphatic groups, are functionalized as ethers, amnines, or the likes, The term nucleicc acid" and "polynucleotide" are used interchangeably herein to describe a polymer of any length, e g,, greater than about 2 bass greater than about 10 5 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, up to about 10,000 or more bases composed of nuelcotides, eggleoxyribonucieotides or ribonucleotides. and maay be produced enzymadcally or synthetically (e,g- PNA as described in U.S. Patent No,948,902 and the references cited therein) which can hybridize with naturally occurring nuclei acids in a sequence specific manner analogous to that of two 10 naturaly occuring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions, Naturalyoccurring nucleotides include guanine, cytosine, adenine and thymine (G, C, A and T respectively), The term "nucleic acid sample," as used herein denotes a sample containing nuceic acid. 15 Tbe term "target polvnucleotide," as used herein., refers to a polynucleotide of interest under study.1i certain embodiments, a target polynucleotide contains one or more target sites that are of Interest under study. The term "igonucleotide" as used herein denotes a single stranded multimer of nucleotides of from about 2 to 200 nucleotides. Oligonucleoides may be synthetic or may be 20 made enzymatically, and, in some embodiments, are 10 to 50 nucleotides in length, Oligonucleouides inay contain ribonucleotide tonoiers (i.e., miay be oligoribonucleotides) or deoxyribonucleotide monomers. An oligonucleotide may be 10 to 20, 11 to 30, 31 to 40, 41 to 50, 51 to 60, 1. to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200 nucleotides in length, for example. 25 The term "duplex" or "duplexedj as used herein, describes two complementary polynuIcle)otides that are base-paired, i.e., hybridized together 'The term "primer" as used herein refers to an oligonucleotide that has a nucleotide sequence that is complementary to a region of a target polynucleodde. A primer binds to the complementary region and is extended, using the target nucleic acid as the template, under 30 priner extension conditions, A primer may be in the nge of about 15 to about 50 nucleotdes although primers uts ide of this length may be used. A priner can be extended from its 3' end by the action of a polymerase An oligonucleotide that cannot be extended from it 3' end by the action of a polyrnerase is not a primer.

The term "extending" as used herein refers to any addition of one or more nucleotides to the end of a nucleic acid, e,g. by ligation of an oligonucleotide or by using a polynerase. The term "amplifying" as used herein refers to generadng one or more copies of a target nuleic acid, using the target nucleic acid as a template 5 The ten "denaturing," as used herin, refers to the separation of a nucleic acid duplex into two single strands. The terms "determining" "measuring",evaluating" "assessing," "assaying" "detecting," and "analyzing" are used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. These terms include 10 both quantitative and/or qualitatikve determinations. Assessing may be relative or absolute. "Assessing the presence of' includes determining the amount of something present, as well as determining whether it is present or absent, The ten "using" has its conventional meaning, and, as such, means employing, e.g putting into service, a method or composition to attain an end. 15 As used herein, the term "T, refers to the melting temperature of an oligonueleotide duplex at which half of the duplexes rmn hybridized and half of the duplexes dissociate into. single strands. The T, of an oligonucleotide duplex may be experimentally determined or predicted using the following formula T = 8 L + 1466(logidNaT]) + 0.41 fractionn G+C (60/N), where N is the chain length and (Na] is less than I M, Sete Sambrook and Russell 20 (200 1 Molecular Cloning: A Laboratory Manual, P ed. Cold Spriag Harbor Press, Cold Spring Harbor N.YL ch 10). Other formulas for Predicting 7 of oligonucleotde duplexes exist and one formula may be more or less appropriate for a given condition or set of conditions, As used herein, the term "Trmatched" refers to a plurality of nucleic acid duplexes 2.5 having T,- that are within a. defined range e g., within 5 "C or 10 "C of each other, As used herein, the terms "reaction mixture" and "reagent mixture" refers to an aqueous mixture of reagents that are capable of reacting together to produce a product in appropriate external conditions over a period of time, A reaction mixture may contain PCR reagents and flap cleavage reagents, for example. 30 The term "mixture", as used herein, refers to a combination of elements, that are interspersed and not in any particular order. A mixture is heterogeneous and not spatially separable into its different constituents. Examples of mixtures of elements include a number of different elements that are dissolved in the sane aqueous solution, or a number of different 4 elements attached to a solid support at random or in no particular order in wbich the different elements are not spatially distinct, A mixture is not addressable. To illustrate by example, an array of spatdally separated surface-bound polynucleotides, as is commonly known in the art., is not a mixture of surface-bound polynucleotides because the species of surface-bound 5 polynucleotides are spatially distinct and the array is addressable. As used herein, the term "PCR reagents" refers to a]l reagents that are required for performing a polyberase chain reaction (PCR) on a template. As is known in the art, PCR reagents essentially include a first primer, a second primer, a thennostable polymerase, and nucleotides. Depending on the polymerase used, ions (e.g, Mg>) may also be present. PCR 10 reagents may optionally contain a template from which a target sequence can be amplified. As used herein, the term "Ilap assay" refers to an assay in which a flap ohgonucleotde is cleaved in an overlap-dependent manner by a fap endonuclease to release a flap that is then detected. The principles of flap assays are well known and described in. esg., Lyamichev et al. (Nat Biotechnol. 1999 -7;292-296), Ryan et al (Mol Diagn, 1999 15 4 135-44)a nd Allawi et al (J (in Microbiol. 2006 44, 3443-3447). For the sake of clarity, certain reagents that are employed in a flap assay are described below. The principles of a flap assay are illustrated in Fig. 1 in the flap assay sh-town in Fig, 1, an invasive oligonucleotide 2 and flap oligonucleotide 4 are hybridized to target 6 to produce a first complex 8 that contains a nucleotide overlap at position 10. First complex 8 is a substrate for 20 flap endonuclease Flap endonuclease 12 cleaves flap oligonucleotide 4 to release a flap 14 that hybridizes with FRET cassette 16 that contains a quencher "Q" and a nearby quenched fourophore "R" that is quenched by the quencher Q. Hybridization of flap 14 to FRET cassette 16 results in a second complex 18 that contains a nucleotide overlap at position 20. The second complex is also a substrate for flap endonuclease, Cleavage of FRET cassette 16 25 by flap endonuclease 12 results in release of the fluorophore 22, which produces a tlorescent signal. These components are described in greater detail below. As used herein, the term "invasive oligonucleotide" refers to an oligonucleotide that is complementary to a region in a target nucleic acid. The 3' terminal nucleotide of the invasive oli gonucleotide may or my not base pair a nucleotide in the target (eg which may be the 30 site of a SNP or a mutation, for example), As used herein, the term "flap oligonucleotide" refers to an oligonucleotide that contains a flap region and a region that is complementary to a region in the target nucleic acidThe target complementary regions on the invasive ligonucleotide and the flap oligonucleotide overlap by a single nucleotide sudh that when they are annealed to the target nucleic acid, the complementary sequences overlap. As is known, if: a) the 3' terminal nucleotide of the invasive nucleotide and h) the nucleotide that overlaps with that nucleotide in the flap oligonuottid both base pair with a nucleotide in the target nucleic acid, then a particular structure is formed, This structure is a substrata for an enzyme, defined below as a 5 flap endonuclease, that cleaves the flap from the target complementary region of the flap oligonucleotide. If the 3' terminal nucleotide of the invasive oligonucleotide does not base pair with a nucleotide in the target nucleic acid, or if the overlap nucleotide in the flap oligononucleotide does not base pair with a nucleotide in the target nudeic acid, the complex is not a substrate for the enzyme and there is little or no cleavage. 10 The term "flap endonuclease" or "FEN" for short, as used herein refers to a class of nucleolytic enzymes that act as structure specific endonucleases on DNA structures with a duplex containing a single stranded 5' overhang, or flap, on one of the strands that is displaced by another strand of nucleic acid, i.e., such that there are overlapping nucleotides at the junction between the single and doubihe-stranded DNA, FINs catalyze hydrolytic 15 cleavage, of the phosphodiester bond at the junction of single and double stranded DNA, releasing the overhang, or the flap; Flap endonucleases are reviewed by Ceska and Savers (Trends Biochem Sci. 1998 23:31-336) and Liu et al (Ann PevBiochei 2004 73: 589 615) .+FNs may be individual enzymes. multi-subunit enzymes, or may exist as an activity of another enzyne or protein complex, e.g, a DNA polymerase, A flap endonuclease may be 20 thermostable As used 'erein, the term "cleaved flap" refers to a si ngle-stranded oligonucleotide that is a cleavage product of a flap assay, As used herein, the term "FRET cassette" refers to a hairpin oligonuceotide that contains a fluorophore moiety and a nearby quencher moiety that quenches the fhuorophore, 25 Hybridization of a cleaved flap with a FRET cassette produces a secondary substrate for the flap endonuclease. Once this substrate is formed, the 5' fluorophore-containing base is cleaved from the cassette, thereby generating a fluorescence signal. As used herein, the term "flap assay reagents" refers to all reagents that are required for performing a flap assay on a substrate. As is known in the art, flap assays include an 30 invasive oigonucleotide, a flap oligonuceotide, a flap endonuclease and a FRET cassette, as described above Flap assay reagents may optionally contain a target to which the invasive oligonucleotide and flap oligonucleotide bind. As used herein. the term genomicc locus" refers to a defined region in. a genome. A genoinic locus exists at the same location in the genomes of different cells from the same 6 individual, or in different individuals, A genonic locus in, one cell or individual may have a nucleotide sequence that is identical or veir similar (ie, more than 99% identical) to the same genomic locus in a different cell or individual, The difference in nucleotide sequence between the same locus in different cells or individuals may be due to one or more nucleotide 5 substitudons A SNP (single nucleotide polymorphism) is one type of point mutation that occurs at the same genomic locus between different individuals in a population, Point mutations may be sornatic in that they occur between different cells in the same individual A genomic locus mutation may be defined by genomic coordinates, by name, or using a symbol. 10 As used herein, a "site ofa mutation" refers to the position of a nucleotide substitution in a genomic locus. Unless otherwise indicated, the site of a nutation in a nucleic acid can have a mutant allele or wild type allele of a mutation. The site of a mutation may be defined by genomic coordinates, or coordinates relative to the start codon of a gene (e.g, in the case of the "KRAS G35T mutation"). 15 As used herein the term "point mutation" refers to the identity of the nucleotide present at a site of a mutation in the mutant copy of a genomic locus. The nucleotide may be on either strand of a double stranded DNA molecule. As used herin the term "wild type", with reference to a genomic locus, refers to the alleges of a locus that contain a wild type sequence. In the case of a locus containing a SNP, 20 the wild type sequence may contain the predominant allele of the SNP, As used herein, the term mutantnt, with reference to a genomic locus, refers to the alleles of a locus that contain a mutant sequence, In the ease of a locus containing a SNP. the mutant sequence may contain a minor alle of the SNP, The mutant allele of a genomic locus may contain a nucleotde substitution that is not silent in that it either alters the expression of 25 a protein or changes the amino acid sequence of a protein, which causes a phenotypic change (e.g., a cancer-melated phenotype) in the cells that are heterozygous or homozygous for the mutant sequence relative to cells containing the wild type sequence. Alternatively, the mutant allle of a genomic locus may contain a nucleotide substitution that is silent. As used herein, the tern' "crrespnnds to" and grammatical equivalens thereof in the 30 context of, for example a nucleodde in an oligonucleotide th at corresponds to a site of a mutation is intended to idemify the nucleotide that is correspondingly- positioned relative to (.e, positioned across from) a site of a mutation when two nuclei acids (e g an oligonucleotide and genomic DNA containing the mutation) are hybridized. Again, unless otherwise indicated (e~g. in the case of a n ucleoide that "does not base pair" or "base pairs" with a point mutation) a nucleotide that corresponds to a site of a mutation may base pair with either the mutant or wild type allele of a sequence. As used herein, the term "KR AS" refers to the human cellular homolog of a transforming gene isolated from the Kirsten rat sarcoma virus, as defined by NCBI's OMIM 5 database entry 190070. A sample that contprises "both wild type copies of the KRAS gene and mutant copies of the KRAS gene" and gmimatical equivalents thereof, refers to a sample that contains multiple DNA molecules of the same genomic locus, where the sample contains both wild type CoJpils of the genomic lucus hichCopies contain the wild type allele of the locus) and 10 mutant copies of the same locus (which copies contain the mutant allele of the locus). In this context, the term "copies" is not intended to mean that the sequences were copied front one another. Rather, the term "copies" in intended to indicate that the sequences are of the same locus in different cells or individuals. As used herein the term "nucleotide sequence* refers to a contiguous sequence of 15 nucleotides in a nucleic acid As would be readily apparent, number of nucleotides in a nucleotide sequence may vary greatly. In particular embodiments, a nucleotide sequence (eg, of an oligonucleotide) may be of a length that is sufficient for hybridization to a complementary nucleotide sequence in another nucleic acid, In these enbodiments, a nucleotide sequence may be in the range of at least 10 to 50 nucleotides, eg, 12 to 20 20 nucleotides in length., although lengths outside of these ranges may be employed in many circumstances, As used herein the term "fully complementary to" in the context of a first nucleic acid that is fully complementary to a second nucleic acid refers to a case when every nucleotide of a coniguous sequence of nucleotides in a first nileic acid base pairs with a complementary 25 nacleotide in a second nucleic acid, As will be described below, a nucleic acid may be fully complementary to another sequence "with the exception of a single base mismatch", meaning that the sequences are otherwise fully complementary with the exception of a single base mismatch (i e, a single nucleotide that does not base pair with the corresponding nucleotide in the other nucleic acid), 30 As used herein the term a "primer pair" is used to refer to two primers that can be employed in a polymerase chain reaction to amplify a genomic locus. A primer pair may in certain circumstances be referred to as containing "a first primer" and "a second primer" or "a forward primer" and "a reverse primer". Use of any of these terms is arbitrary and is not 8 intended to indicate whether a primer hybridizes to a top strand or bottom strand of a double stranded nucleic acidL The nucleotides of an oligonucleotide may be designated by their position relative to the 3' terminal nucleotide of an oligonucleotide, For example the nucleotide immediately 5' 5 to the Y terminal nucleotide of an oligonucleotide is at the "-1" position the nucleotide immediately 5' to the nuleotide at the -i position is the "-2" nucleotide, and so on. Nucleotides that are "within 6 bases" of a 3' terminal nucleotide are at the -1, -2. -3, -4, -5 and -6 positions restive to the 3' terminal nucleotide, 10 DESCRIPTION OF EXEMPLARY EMBODIMENTS Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is al-so to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of 15 the present invention will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed wthin the invention. The upper and lower limits of these smaller 20 ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same 25 meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials sindiar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and nateri a are now described, All publications and patents cited in this specification are herein incorporated by 30 reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.he citation of any publication is for its disclosure prior to the fiing date and should not be construed as an admission that the present invention is not entitled to antedate such 9 publication by virtue of prio invention, Further, the dates of publication provided may be different from the actual publication dates which may need to he independently confirmed. It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise It is 5 further noted that the claims may be drafted to exclude any options eiemen As such. this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely,"' "only' and the like in connection with the recitation of claim elements, or use of a "negative" limitation. As will be apparent to those of skill in the art upon reading this disclosure, each of the 10 individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. 15 In the following description, the skilled artisan will understand that any of a number of polymerases and flap endonucleases could be used in the methods. including without linitation, those isolated from thermostabhe or hyperthermostable prokaryotic, eukaryotic, or archacal organisms, The skilled artisan will also undersuand that the enzymes that are used in the method, e~g, polymerase and flap endonuclease include not only naturally occurring 20 enzymes, but also recombinant enzymes that include enzymatically active fragments, cleavage products, mutants, and variants of wild type enzymes. In further describing the method, the reagent mixture used in the method will be described first, followed by a description of the reaction conditions that may be used in the method, 25 Reagent mixtures A reagent mixture is provided. In certain embodiments, the reagent mixture comprises: a) amplification reagents comprising a thermostable polymerase, micleotides, a set of at least seven forward pritner and a reverse primer, wherein: i. the 3' terminal nucleotide 30 of ea forward primer of the set base paih with a different point mutation in the KR AS gene relate t other forward primers in the set, v hhrein t point mutation is selected from the following point ra-ations:34A, 34C 341 35A 35C, 35T and 38A; ii. each of the forward primers comprises a nucleotide sequence that is fully complementary to a sequence in the KRAS gone with the exception of a single base mismatch within 6 bases of the 3' terminal I ) nucleotide; and iii. each of the forward primers, in combination with the reverse priner selectively amplifies a different allele of a KRAS gene, wherein the allele that is amplified is defined by the point mutation to which the 3' terminal nucleotide base pairs; and b) flap assay reagents comprising a flap endonuclease a first FRET cassette that produces a fluorescent 5 signal when cleaved, the set of at least seven forward primers, and a corresponding set of at least seven different flap oligonucleotides that each comprise a nucleotide that base pairs with one of the point mutationst wherein the reagent mixture is characterized in that, when the reagent mixture combined with a nucleic acid sample that comprises at least a 1.000-fold excess of wild type copies of the KRAS gene relative to mutant copies of the KRAS gene that 1.0 contain one of the point mutations and thermocycled, the reagent mixture can amplify and detect the presence of the mutant copies of the KRAS gene in the sample. The reacton mixture is characterized in that it can. amplify and detect the presence of mutant copies of the KRAS gene in the sample. The forward primers of the amplification reagents are employed as an invasive primer in the flap assay reagents, 5 'The exact identities and concentrations of the reagents present in the reaction mixture may vary greatly but may be simlar to or the same as those independently employed in PCR and flap cleavage assays, withe exception that the reaction mixuMre. may contain Mg't a concentration that is higher than employed in conventional PCR reaction mtixtures (which contain Mg 2 at a concentration of between about 18 mM and 3 mM). In certain 20 rbodimen ts, the reaction mixture described herein contains Mg at a concentration of in the range of 4 mM to 10 mM, e-g., 6 mM to 9 mM- Exemplary reaction buffers and DNA polymerases that may be employed in the subject reaction mixture include those described in various publications (e.g., Ausubel. et al. Short Protocols in Molecular Biology, 3rd ed . Wiley & Sons 1995 and Sambrook et al,, Molecular Cloning: A Laboratory Manual, Third 25 Edition, 2001 Cold Spring Harbor, N.Y.) Reaction buffers and DNA polymenrases suitable for PCR may be purchased from a variety of suppliers, e.g., Invitrogen (Carlsbad, CA), Qiagen (Valencia, CA) and Stratagene (La Jolla, CA), Exemphry polymerases include Taq, Pfu, Pwa Uilana and Vent. although many other polymerase may be employed in certain embodiments. Guidance for the reaction components suitable for use with a polymerase as 30 well as suitable conditions for their use is found in the literature supplied with the polymerase. Primer design is described in a vanety of publications, eg., Diffenbach and Dveksler (PCR rimer, A Laboratory Manual, Cold Spring Harbor Press 1995), . Rapley, (The Nucleic Acid Protocols Handbook (2000) Humana Press, Totowa, NJ); Schena and Kwok et al., NucL Acid Res. 1990 18'999-1005) Primer and probe design software programs 11 are also commercially available, including without initadion, Primer Detective (CionTech, Palo Alto, Calif.), Lasergene (DNASTAR, Inc., Madison, Wis..), and Oligo software (National Biosciences, Inc., Plymouth, Minn) and iCligo (Caesar Software, Portsmouth, N.H), S Exemplairy flap cleavage assay reagents are found in Lyaznichev et al. (Nat, Biotechnol, 1999 17:292-296), Ryan et al (Mol Diagn. 1999 4;135-44) and Aliawi et al Q Clin Microbiol 2006 44: 3443-3447). Appropriate conditions for flap endonuclease reactions are either known or can be readily determined using methods known in the art (see, e.g., Kaiser et al., J Biol. Chem. 274:21387-94, 1999), Exemplary flap endonucleases that 10 may be used in the method include, without limitation, Thermus aquaticus DNA polymerase I, Thernutrmphii DN A pol ymerase . manmmalian FENi, Archaeoglobusfulgidus FEN-I, Methanococcus jannaschii FN I Pyrococcusfuriosus FEN- IMethanbacter an thermoautotrophicwn FEN-, Theermophius FEN-i CLEAVASE (Third Wave, Inc., Madison, Wis.), S. cerevisiae RTHI S, cerevisiae RAD27, Schizosaccharomyces pombe 15 rad2 bacteriophage S5-3'exonuease. Pyroccus horikoshii FEN-I, human exonuclease 1, calf thymus 5'-I exonuclease, including homologs thereof in eubacteria, eukarvotes, and archaea, such as members of the class II family of structure-specific enzymes, as Well as enzymatically active mutants or variants thereof Descriptions of cleaving enzymes can he found in, among other places, Lyamichev et al., Science 260:7883, 1993; Eis et al, Nat. 20 Biotechnol 19:673-76, 2001; Shen et al, Trends in Bio, Sci 23:171-73, 1.998; Kaisereet al, J. siol. Chem. 274:1387-4 999; Ma et al., J. Biol, Chem. 275:24693-700, 2000; Allawi et al,, J. Mol. BioL 33 537-54, 2003; Sharma et alJ. Bi. Chem. 278:23487-96, 2003; and Feng et al Nat Struct Mol, Biol. 11:450-56, 2004. As noted abovo, the reaction mix contains reagems for assaying for, in a single vessel, 25L seven different targets mutations in the KRAS gene. As such, the reaction mix contains multiple forward primers (the 3' bases of each of which base pairs with one of the seven point mutations), a single reverse primer, multiple different flap oligonucotides that each have a nucleotide that base pairs with a singe point mutation, and at least one FET cassette for detecting flap cleavage In one embodiment flap olgonucleotides in aiixture may have 30 a common flap to allow for, for example, the production of the same single fluorescem signal if any of the seven flap oligonuclcotides is cleaved, In another embodiment, the flap assay reagents comprise a first FRI .1 cassette and a second FRET cassette that produce distinguishable fluorescent signals when cleaved, and at least one of the at least seven different flap ogonucleodesomprises a flap sequence that hybridizes to the first FRET cassette and the remainder of said at least seven different flap oligonucleotides hybridizes to the second FR ET cassette. In these embodimems, one fluorescent signal will indicate that one of the subset of the mutations is present, whereas the otner fluorescent signal will indicate that one of the other mutations is present., 5 In certain cases the reagent mixture may contain a PCR primer pair, a flap oligonuclentide and FRETr cassette for the detection of an internal control. In these embodiments, the reaction mixture may further comprise second amplification reagents and second flap reagents for amplifying and detecting a control sequence that is in a gene that is not in KRASI wherein said second flap reagents comprise a second FRET cassette that 10 produces a signal that is distinguishable from the signal of the first FRET cassette, In particular cases, the control gene may be D-actin. although any suitable sequence may be used. Upon cleavage of the FRET cassettes, multiple distinguishable fluorescent signals may be observed. The fluorophore may be selected from, eg 6-carboxyfluorescein (FAM), 15 which has excitation and emission wavelengths of 485 in and 520 nm respectively, Redmond Red, which has excitation and emission wavelengths of 578 mu and 650 nm respectively and Yakima Yellow, which has excitation and emission wavelengths of 532 am and 569 nm respectively, and Quasor670 which has excitation and emission wavelengths of 644 nm and 670 nm respectively, although many others could be employed. 20 As noted above, seven of the PCR primers (arbitrarily designated as the "forward" primers). compnses a 3' tenninal nucleotide that base pairs with a point mutation (ie., a nmutant allele) mn the genomic locus and also comprises a nucleotide sequence that is fully complementary to a sequence in the locus with the exception of a single base mismatch within 6 bases of the 3 terminal nucleotide (e.g., at the -p the -2 position, the -3 25 position, the -4 position, the -5 position or the -6 position, relatve to the 3' terminal nucleotide). In other words, in addition to having a IT tenninal nucleoide that base pairs with only the mutant allele of the mutation in the genomic locus, the priner also has a destabilizing mismatch near the 3' end that neither bases pairs with the mutant allele or the wild type allele of the genomic region. The mismatch may be at the same or different 30 positions in each of the forward primers. Without being limited to any particular theory, the destabilizing mismatch is believed to destabilize hybridization of the 3' end of the first primer to the wild-type sequence to a greater extend than mutant sequence, thereby resulting in preferential amplification of the mutant sequence. As will be described in greater detail below, the presence of the product amplified using the first and second primers may be 13 detected using a flap ass'ay that employs the first primer or another oigonucleotide that has the destabilizing mutation and a. terminal nucleotide that base pairs with only the mutant allele at the genomic locus. The use of such a sequence (ie, a sequence that contains the destabilizing mutation and a terminal nucleotide that base pairs with only the mutant allele at 5 the genonic locus) in the detection step provides further discrimination between mutant and wild type sequences in the amplification products, Without being bound to any particular theory, it is believed that the discrimination between mtant and wild type largely occurs in the first few rounds of amplification since the ampified sequence (i.e., the amplicon) provides a perfectly complementary sequence for the PCR primers to hybrdize to. The wild 10 type sequence should not be amplified, whereas the mutant sequence should be efficiently amplified. The length. of the nucleotide sequence that is complementary to the KR..A.S gene in the forward primers may be at least 16 nucleotides in length (e.g, at least 17 nucleotides, at least 18 nuc'leotides. at least 19) nuicleotides, to at least 10 nucleotides or more, in length). The destabilizing mismatch can be done by substituting a nucleotide that base pairs 15 with the point mutation with another nucleotideThe nucleotide that is substituted into the sequence may be another natural nucleotide (e.g., dG, dA, dT or dC), or, in certain circumstances, a modified nucleotide. In certain embodiments, the 3' end of the first primer may contain more than l e 2 or 3, mismatches. In particular embodiments. the type of mismatch (eg, whether the mismatch is a G:T mismatch or a CT mismatch, etc ) used 20 affects a primer's ability to discriminate between wild type and mutant sequences. In general terms, the order of the stabilities (from most stable to least stable) of various mismatches are as follows: G:TF > CT: = A: C > T:G > G:A =T:T > TC > A:C > C:T> A:A > C:A > C:C (as described in Gaffney and Jones (Biochemistry 1989 26: 5881-5889)), although the basepairs that surround the ismatch can affect this order in certain circumstances (see. e.g, Ke et al 25 Nucleic Acids Res. 1993 21:5 13 I7- 51 43).The mismatch used may be optimized experimentally to provide the desired discrimination. As would be apparent, the various oligonucleotdes used in the method are designed so as to not interfere with each other. For example, in particular embodiments, the flap oligonucleotide may be capped at its 3' end, thereby preventing its extension. Further, in 30 certain cases, the TNs of tne flap portion of the flap oligonucleotide and the target complementary regions of the flap oligonucleotide may independently be at least 1 OC lower (e.g_ 10-20 T lower) than the. T,-, of the PCR primes, which results in a) less hybridization of the flap oligonucleotide to the target nucleic acid at higher temperattes (65 C to 75 "C) and h) less hybridization of any cleaved flap to the FRET cassette at higher temperatures (65 14 "C to 75 "C), thereby allowing the genomic locus to be amplified by PCR at a temperature at Which the flap does not efficiently hybridize In. particular cases, the forward prners used for detection of the KRAS mutations may haye at least 12 contiguous nucleotides (e.g at least 13, 14, 15, 16, 17 or 18 contiguous 5 nucleotides) starting from the 3' end of thefollowing sequences: ACTTGTGOCTAGTTOGACCWA (SEQ ID NO: 1), ACTTGTGGTAGTTOOAGCTCT (SEQ ID NO: 2), AACTTGTOG3TAGT'IGGAGATGC (SEQ ID NO: 3), CT'TGTOGITAOTT'clGAGOCcA (SEQ ID NO: 4), CTTGTGGTAGTTG3GAGCCT (SEQ ID NO: 5), TATAAACTTOTOGTAOTTGOACCTC (SEQ ID NO: 6), 10 TGGTAGTTGGAOCTGOIAA (SEQ ID NC: 7) The flap probe may in certain cases base pair with 10 to 14 contiguous nucleotides, eg., 11. to 13 contiguous nucleotides, of the KRAS gene, in a multiplex reaction, the primers may be designed to have similar thenrodynamic properties, e.g, similar 7s, G/C content, hairpin stability, and in certain embodiments may 15 all be of a similar length, e, from 18 to 30 nte.g., 20 to 25 nt in length. The other reagents used in the reaction mixture may also be 7, matched, The assay mixture may be present in a vessel, including without limitation, a tube; a multi-well plate, such as a 96-well, a 384-wella 5 plate; and a microfluidic device. In certain enbodiments, multiple multiplex reactions are performed in the same reaction 20 vessel Depending on how the reaction is performed, the reaction mixture may be of a volume of 5 pt to 200 U. e.g." 10 d to 100 although volumes outside of this range are envisioned. In certain embodiments, subject reaction mix may further contain a nucleic acid sample. In particular embodiments, the sample may contain genomic DNA or an amplified version thereof (e .g. genomic DNA amplified using the methods of Lage et al, Genome Res 25 2003 13: 294-307 or published patent application US20040241658, for example). In exemplary embodi ents, the genomic sample may contain genomic DNA from a mammalian cell, such as, a human, mouse rat, or monkey cell. The sample may be made from cultured cells or cells of a clinical samples eg. a tissue biopsy, scrape or lavage or cell of a forensic sample (1e, cells of a sample collected at a crime scene). In paf ticular embodiments, the 30 genonic sample may be from a formalin fixed paraffin embedded (1TPE) sample. In particular ebodimenCt the nucleic acid sample may be obtained from a biological sample such as cells tissues, bodily fluids, and stool, Bodxily fluids of interest include but are not limited to, blood, serum, plasma, saliva, mucous, phlegm, cerebral spinal fluid, pleural fluid, tears. lactal duct fluid, lymph, sputur, cerebrospinal fluid, synovial fluid, urine, is amniotic fluidand semen. In particular embodiments, a sample may be obtained from a subject, e.g., a human, and it may be processed prior to use in the subject assay, For example the nucleic acid may he extracted from the sample prior to use, methods for which are known. For example, DNA can be extracted from stool from any number of different 5 methods, including those described in, e.g. Coll et al (T. of Clinical Microbiology 1989 27: 2245 -2248), Sidransky et al (Science 1992 256: 102-105)j Villa (Gastroenterology 1996 110: 1.346-1353) and Nollau (ioTechniques 1996 20:784788), and U.S, Patents 5463782, 7005266, 6303304 and 5741650. COommercial DNA extraction kits for the extraction of DNA from stool ielude the QiAamp stool mini kit (QIAGEN Hilden, Germany), Instagene 10 Matrix (i- Rad. Hlerdculs Cli s ) and RapidPrep Micro Genomic DNA isolation kit (IPharmacia otech ine iscat way. N ), among other Aehodfor sawple analysis A method of sample analysis that employs th e reagent mix is also provided, In certain 15 embodiments, this method comprises: al subjecting a action mixture comprising i. the above-summarized reagent mixture and ii. a nucleic acid sample that comprises at least a 100-fold excess of wild type copies of the KRAS gene relative to mutant KRAS gene that contain one of the point mutations, to the following thermocycling conditions: a first set of 5 15 cycles of: i. a first temperature of at least 90 TC; i. a second temperature in the range of 60 20 "C to 75 0"C; iiil a third temperature in the range of 65 T to 75 T; followed by: a second set of 20-50 cycles of: i. a fourth temperature of at least 90 ", ii, a fifth temperature that is at least 10 " lower than the second temperature; iii. a sixth temperatum in the range of 65 "C to 75 TC wherein no additionaJ. reagents are added to the reaction between the first and second sets of cycles and, in each cycle of the second set of cycles cleavage of a flap probe is 25 measured; and b) detecting the presence of a mutant copy of KRAS in the nucleic acid sample. in these embodimients the reaction mixture may be subject to cycling conditions in which an increase in the. amount of amplified product (indicated by the amount of fluorescence) can be measured in real-time, where the term eal me is intended to refer to 30 a measurement that is taken as the reaction progresses and products accumulate. The measurement may be expressed as an absolute number of copies or a relative amount when normal zed to a control nucleic acid in the sample. Fluorescence, can be monitored in each cycle to provide a real tine measurement of the amount of product that is accumulating in the reaction mixture. 16 In some embodiments, the reaction mixture may be subjected to the following thermocycling conditions: a first set of 5 to 15 (e g., 8 to 12) cycles of: i. a first temperature of at lea"t 90 0 C; ii, a second temperature in the range of 60 C to 75 (e.g , 65 "Cto 75 "C iii a third temperature in the range of 65 "C to 75 'C: followed by' a second set of 20-50 5 cycles of: i. a fourth tempeature of at least 90 "C; ii. a fifth temperature that is at least 10 "C lower than the second temperature (e.g" in the range of 50 C to 55 "C); and iii, a sixth temperature in the range of 65 'C to 75 "C No additional agents need to be added to the reaction mixture during the thermocycling, eg between the first and second sets of cycles. In p rticuiar embodiments, the thermostable polymeTase is not inactivated between the first 10 and second sets of conditions, thereby allowing the target to be amplified during each cycle of the second s of Cycles. In particular embodiments, the second and third temperatures are the same temperature such that "two step" thernocycling conditions are performed, Each of the cycles may be independently of a duration in the range of 10 seconds to 3 minutes, although. durations outside of this range are readily employed. In each cycle of the second set 15 of cycles (eg., while the reaction is in the fifth temperature), a signal generated by cleavage of the flap probe may be measured to provide a real-time measurement of the amount of target nucleic acid in the sample. The method provided herein is a multiplexed invader assay that employs mismatched pri mers The subject method may be readily adapted from the method shown in Fig, 2 by the 20 addition of at least six other pdri.ners that recognize other point mutations in the KRAS gene, as described above. With reference to Fig. 2, the method includes amplifying product 30 from sample 32 that comprises both wd type copies of the KRAS gene 34 and mutant copies of the KRAS gene 36 that have a point mutation 38 (e-g. the 34A, 34,, 34T, 35A, 35C, 35T or 38A mutations) relative to the wild type gene 34, to produce an amplified sample The 25 amplifying is done using a forward primner 40 and a second primer 42, where the first primer c..omnprises a ' terminal nucleotide 44 that base pairs with the point mutation and also comprises a nucleoide sequence that is fully complementary to a sequence in the locus with the exception of a single ease mismatch 46 (i.e., a base that is not complementary to the corresponding base in the target genomic locus) within 6 bases of 3* terinal nucleotide 44. 30 The presence of product 30 in the amplified sample is detected using a flap assay that employs the same forward primer as an invasive oigonucleotide 48 As shown in Fig. 2, the forward primer 40 is employed as the invasive oligonucleotide 48 in the flap assay. As described above and in Fig. 1, the flap assay relies on the cleavage of complex 32 that contains a flap 0liigonucleotide 50, invasive oligonuceotde 48 and product 30 by a flap 1,7 endonuelease (not shown) to release flap 52. Released flap 52 then hybridizes to FRET cassette 54 to form a second complex that is cleaved by the flap endonuclease to cleave the fluorophore from the complex and generate fluorescent signal 56 that can be measured to indicate the amount of product in he amplifed sample. In this embodiment, the presence of a 5 fluorescent signal indicates that there are mutant alleles of the KRAS gene in the sample. The amount of product in the sample may be normalized relative to the amount of a control nucleic acid present in the sample, thereby determining a relative amount of the notant copies of KRAS in the sample. In some embodiments, the control nucleic acid may be a different locus to the genomic locus and, in certain cases, may be detected using a flap 10 assay that employs an invasive oligonucleotide having a 3 terminal nuCleotide that base pairs with Lhe wild type copies of the genomic locus at the site of the point nutation, thereby detecting the presence of wild type copies of the genomic locus in said sample. The control may be measured in parallel with measuring the product in the same reaction mixture or a different reaction mix. If the control is measured in the same reaction mixture, the flap assay 1.5 may include further reagents, particularly a second invasive oligonucleotideasecond flap probe having a second lap and a second FRET cassette that produces a signal that is distinguishable from the FRET cassette used to detect the mutant sequence, in particular embodiments, the reaction mixture may further comprise PCR reagents and flap reagents for amplifying and detecting a second genonmic locus or for detecting a second point mutation in 20 the same genomic locus. in certain cases, fluorescence indicating the amount of cleaved flap can be detected by an automated fluoronieter designed to perform realtime PCR having the following features: a light source for exciting the fluorophore of the FRET cassette, a system for heating and cooling reaction mixtures and a fluorometer for measuring fluorescence by the FRET 25 cassette. This combination of features, allows real-time measurement of the cleaved flap, thereby allowing the. amount of target nucleic acid in the sample to be quanti fied. Automated fluoromueters for performing real-time lFCR reactions are known in the art and can be adapted for use in this specific assay, for example the ICYCLERTM from Bio-Rad Laboratories (Hercules, Calf, the Mx3000Pt the MX3 005P and the MX4000TM from Stratagene (La 30 Jolla. Calif' the ABI PRTSNMr 7300, 7500, 7700, and 7900 Tag Man (Applied lisystems, Foster City, Cali), the SMARTCYCLERTN, ROTORGIIENE 2000N1 (Corbett Research, SydneyAustralia) the GENE XPERT'M Systerm (Cepheid, Sunnyvale, Calif.) and the LIUGHTCYCLERTM (Roche Diagnostics Corp., Indianapolis, Ind). The speed of ramping is between the different reaction temperatures is not critical and., in certain embodiments, the default ramping speeds that are prese t on thermocyclers may be employed. In certain eases, the method may further involve graphing the amount of cleavage that occurs in several cycles, thereby providing a real time estimate of the abundance of the 5 nucleic acid target. The estimate may be calculated by determining the threshold cycle (i. the cycle at which this fluorescence increases above a predetermined threshold; the "t" value or "Cp" value), This estimate can be compared to a control (which control may be assayed in the same reaction mix as the genomic locus of interest) to provide a normalized estimate. The thermocycler may also contain a software application for detennining the 10 threshold cycle for each of the samples. An etxenplary method for determining the threshold cycle is set forth in, egO, Luu-The et a] (Biotechniques 2005 38; 28 .93), A device for performing sample ana ysis is also provided In certain embodiments, the device comprises: a) a thermocycler programmed to perform the above-deseibed method and b) a vessel comprising the above-described reaction mixture. 15 Kits Also provided are kits for practicing the subject method, as described above. The components of the kit may be present in. separate containers, or multiple components may be present in a single container. In particular embodiments, a kit may comprise: a) amplification 20 reagents composing a thermostable polymerase, nucleotides a set of at least seven forward primers, and a reverse primer, wherein: i, the 1' terinal nucleotide of each forward primer of the set base pairs with a different point mutation in the KRAS gene relative to other forward primers in the set, wherein the point mutation is selected from the following point stations: 34A, 34 34T, 35A, 35C, 35T and 38A, ii. each of the forward primers 25 comprises a nucleotide sequence that is fully complementary o a sequence in the KRAS gene with the exception of a single base mismatch within 6 bases of the 3' terminal nucleodde; and iii. each of the forward primers in combination with the reverse primer, selectively amplifies a different allele of a KRAS gene, wherein the allele that is amplified is defined by the point mutation to which the 3 tLminal nucleotide base pairs and b) flap assay reagents 30 comprising a flap endonuclease, a FRET cassete, the set of at least seven forward primers, and a corresponding set of at least seven different flap oligonucleotides that each comprise a nucleotide that base pairs with one of the point mutations. The particulars of these reag;e;nts are described above The kit further comprises PCR and flap reagents for amplification and detecdon of a control nucleic acid. 19l In addition to above-mentioned components. the kit may further include instructions for using the components of the kit to practice the subject methods. The instructions for Practicing the subject methods are generally recorded on a suitable recording medium. For example. th e instructions may be printed on a substrate, such as paper or plastic etc. As such, 5 the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i-e., associated with the packaging or subpackaging) etc, In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e, g CD-ROM. diskette, etc. In yet other eLmbodiments, the actual tnstructiuns are not present in the kit, but means for obtaining the 10 instructions from a remote source. e.g. via the internet, are provided. An example of this enbodimem is a kit that includes a web address where the instoructions can be viewed and/or from which the instructions can be downloaded As with the instructons, this means for obtaining the instructions is recorded on a suitable substrate. In addition to the instructions, the kits may also include one or more control samples, e g., positive or negative controls 15 analytes for use in testing the kit Utility The method described finds use in a variety of applications, where such applications generally include sample analysis applications in which the presence of a mutant KRAS gene 20 in a given sample is detected. In particular, the above-described methods may be employed to diagnose, to predict a response to treatment, or to investigate a cancerous condition or another mammalian disease, including but not limited to, a variety of cancers such as lung adenocarcinoma, mucinous adenoma. ductal carcinoma of the pancreas and colorectal carcinoma, Noonan syndrome, bladder cancer, gastric cancer. cardio-facio-eutaneous 25 syndrome, leukemias, colon cancer, pancreatic cancer and. lung cancer, for example In some embodiments, a biological sample may be obtained from a patient, and the sample may be analyzed using the method. tn particular miments, the method may be employed to identify and/or estimate the amount of mutant copies of a genomic locus that are in a biological sample that contains both wild type copies of a genomic locus and mutant 30 copies of the genomic locus that have a point mutation relative to the wild type copies of the genomnic locus In this example, the sample may contain at least 100 times (eg., at least 1,00 times, at least 5,000 times. at least 10,000 times, at least 50.0()0 times or at least 100,000 tines) more wild type copies of the KRAS gene than mutant copies of the KRAS gene. 20 Since the point mutation in the KRAS gene have a direct association with cancere, colorectal cancer, the subject method may be employed to diagnose patients with cancer or a pre-cancerous condition (e.g., adenoma etc.), alone, or in combination with other clinical techniques (e g, a physical examination, such as, a colonoscopy) or molecular techniques 5 (eg., immunnhistotbemical analysis. For example, results obtained from the subject assay may be combined with other information, e.g. information regarding the methylation status of other loci, information regarding rearrangements or substitutions in the same locus or at a different locus, cytogenetic information, information regarding expression information or information about the length of telomeres, to provide an overall 10 diagnosis of cancer or other diseases. In additional eubodiments, if a KRAS mutation is detected in a sample, the identity of the mutation in the sample may be determined. This may be done by, e.g., sequencing part of the KRAS locus in the sample, or by performing seven separate assays (i.e. using the same reagecnts, but not in muliplex form) to determine which of the stations is present. 15 In one embodiment, a sample may be collected from a patient at a firs location, e.g,, in a clinical setting such as in a hospital or at a doctor's office, and the sample may be forwarded to a second locationeg.. a laboratory where it is processed and the above described method is performed to generate a report, A "report' as described herein, is an electronic or tangible document which includes report elements that provide test results that 20 may include a Ct value, or Cp value, or the like that indicates the presence of rutant copies of the genomic locus in the sample. Once generated, the repon may be forvarded to another location (which may the same location as the first location), where it may be interpreted by a health professional (e.g, a clinician, a laboratory technician, or a physician such as an oncologist surgeon, pathologist), as part of a clinical diagnosis All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference The citation of any publication is for its disclosure prior to the filing date and should not be constmed as an 30 admission that the present invention is not entitled to antedate such pubacaton by virtue of prior invention. Although the foregoing invention has been described in some detail by way of Illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes 21 and modifications may be made thereto without departing from the spirit or scope of the appended claims. EXAMPLE 1 5 MATERIALS AND METHODS Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States, yet whit effective screening it is potentially the most treatable and preventable cancer JNCI201.0102:89, Ann Intern Med 2009150:1 Ann Intern Med 2008149:441), 10 The aim of this study was to evaluate the performance of the mutation detection component of an assay by testing a set of colorectal tissues that were characterized using standard dideoxynucleotide sequencing. The assay is designed to detect the common KRAS mutation sequences at Codons 12 and 13, which are found in approximately 35% of all colorectal cancer tissues, The current assay combines all seven KR AS mutations in a sigle 15 reaction. ACTB1 (beta-acin) is also included in the reaction to confirm sufficient DNA levels and to ratio KRAS against to establish percent mutation. A mutiplexed KRAS assay was designed utilizing QuARTS (Quantitative Allele specific Real ime Target and Signal amplification), a highly sensitive technology that combines alele-specific DNA amplification with invasive cleavage chemistry to generate 20 signal during each amplification cycle similar to real-time PCR. The assay, which detects seven KRA S mu t ations and the reference gene R-acuin. was used to assess 87 colorectal tissue samples (52 CRCs, 16 adenomas > lcm, and 19 normal epithelia) as determined by Mayo Clinic Pathology, Samples were obtained by microdissection of fresh frozen tissue biopsies. DNA was extracted by Mayo Clinic using a standardized chloroform/pheno methodology 25 The genotypes of each sample were established using dye terminator dideoxynucleotide sequencing in both the forward and reverse orientations Copy numbers of KAS mutations andfi-acian were determined by conventional comparison against standard curves. KRAS data are reported as percent mutation and calculated by dividing mutant copies by fl-acuin copies and multiplying by 100, 30 Tissue Sample excising, extraction, and sequencing Tissue samples were collected from adenoma and primary tumors and normal colons at the Mayo Clinic with IRB approval, Patients with confirmed neoplasia had been identified by colonoscopy, endoscopy, radiologic, and/or ultrasound studies, Normal colonic tissue samples were collected from colonoscopy negative patients. For the tunors, pathologist 22 examined the issue sections and circled out histological1y distinct lesions to direct careful micro-dissection with about 80% purity. DNA was extracted at Mayo Clinic using either the QlAamp DNA Micro Kit (PN 56304 Germantown, MD) or a standardized chloroforn/phenol methodology. Tissue DNAs were stored at -80Ct 5 Sequencina The KRAS genotypes of each cancer or adenoma sample were established using dye terminator dideoxynucleotide sequencing in both the forward and reverse orientations for a region including codons 12 and 13 of the KRAS gene, Samples were sequenced on an ABI 3730XL DNA Analyzer using Big Dye Terninator v3. I reagents (Applied Biosystens) 10 Mutation Surveyor v,30 software (SoftGenetics) was used to make the calls. Lane quality scores for the traces were greater than 20, indicating less than 5% average background noise, and base calls were made based on signal to noise ratios, peak heights, overlap, and drop-off rate, When quality scores were above 20 in both directions, concurrence in both directions was required to verify an alteration rorn wild type (WT) if one direction was of low quality., 15 but the other was above the threshold of 20, the single high quality read was sufficient to make a call Traces were manually inspected for accuracy and 2'. positive calls were made which were below the sensitivity of the software, Normal colon samples were not sequenced. Only mutation 34C was notrepresented in these samples. QuARTS Assay Techniques 20 DNA samples extracted from tissues were assessed for the presence of mutations in exon 2 of the KRAS gene (See Table 1) and the reference gene Beta-Actin using a multiplexed QuARTS (Quantitative Allele-specific Real-time Target and Signal amplification) assay, 25 Table 1 KR AS< RAS Codon Amino Amino Amino Mmao utation nationn ~acid in I acid acid ih sho no: . ttation change KR AS ----- r--- .a ..... . W T 35G>A____ 35A ___ Codon I' Aspartate | Gly2Asp 35G>T 35T - -Codon 12 \Valin | Gly 1Val 34G>T 34T odo n 12 Cstine | Ly12Cys 35G>C 3C Codon 12 Ala nine L G ly12Ala Glycine 34G>A 34A Codon 12 Serine Gly 12Ser 34 C 34C Codon 12 Arginine y G 2Arg 38C)>A 38A Codo n 1.3 Aspartate GIl3As ______p 23 The assay is specific to the mutant KRAS DNA and is able to discriminate mutants from wild-type with low cross-eactivity Specificity is achieved by the use of allele specific PCR with specific mismatches in the forward primer to prcferttily amplify mutant alleles combined with semi-quantitative invasive cleavage reactions that further discriminate and 5 detect the amplified target using real-tme fluorescence dtckcion, The QuA RTS reaction was optimized so the primers and probes for each mutation would function properly at same cycling and reaction conditions allowing all eight markers to be scorned mna single reaction. Cycling conditions are designed to preferentially amplify mutant sequences by using a higher annealing temperature in the first 10 cycles, followed by 10 35 cycles at lower annealing temperature required for the invasive cleavage reaction, Fluorescent acquisition begins after the first 10 cycles, Multiplex KRAS assays were first Opirnized in a two-dye configuration where all mutations reported to one dye while ACTB reported to a second dye. T'he assay was further optimized to improve specificity and sensitivity by moving to a 3-dye configuration. The 3-dye KRAS QuARTS muliplex is 15 configured to report to different dyes so that 4 mutations report to one dye (G35A, G35C, 0G34A, and 034C) 3 report to a second dye (G35T, G341 and G38A), and ACTB reports to a third dye (see Figure 3), The KRAS QuARTS multiplex technology generates highly sensitive and specific signal from mutant KRAS sequences and a beta-actin. reference gene by utilizing two 20 simultaneous reactions (Figure 3). In the first reaction, alelespecific amplification is achieved with a unique forward primer for each mutation in combination with a single KRAS reverse prnier Each forward primer contains a double mismatch to tie KRAS WT sequence near the 3 end of the primer, which prevents efficient amplification of KRAS WiT, but has only a single mismatch to the mutation sequence. Taq (recombinant Hot Start Go Taq, 25 Promega, Madison, WY) is able to extend efficiently through a single mismatch but not through a double mismatch near the 3'end. Signal generation occurs in the second reaction. A target specific probe binds to the mutant amplicon to form an overlap flap substrate The 5' flap is then cleaved by the Cleavase enzyme (Hologie, Madison, WI). 'The flap sequence is complementary to a FRET cassette, Once the flap is cleaved, it binds to the target FRET 30 cassette and causes the release of the fluorophore to generate signal. The seven KRAS probes share two different flap sequences, which report to either a HEX or FAM FRET cassette. A probe specific to beta-actin contains a third flap sequence and reports to Quasar 670 FRET cassette. The use of 3 different flap. sequences that correspond to a FAM, HEX or Quasar 670 FRET cassette allows the assay to distinguish KR AS mutations from the beta-actin reference 24 gene in a single well. In total. the KRAS multiplex QuARTS assay combines seven KRAS forward primers, a single KR A S reverse primer, 7 KRAS probes, a beta-actin forward and reverse primer, and three FRET cassettes. The concentration ranges for primers are from 105 to 245 nM, probes are from 90 to 250 aM, and FRET cassettes are at 100 nM each. 5 Reactions were setup by adding 10 pli of DNA from each sample to appropriate wells of a 96-well plate containing 20 gL. of assay-specific QuA.RT'S'master mix. Each plate was mn on an ABI 7500 Fast Dx Rea Time PCR instrument. Calibrators and controls were included in each run. After the run was completed, data was exported to an Exact Sciences analysis template, and the cycle threshold value was calculated as the cycle at which the 10 fluorescent signal per channel for a reaction crosses a threshold of 18% of the maximum fluorescence for that channel, DNA strand number was determined by comparing the cycle threshold of the target gene to the calibrator curve for that assay. Calibrators were made from plasimids with single target inserts, mutation 38A was used for the FAM channel and 35C was used for the HEX channel. Percent mutation was determined for each marker by dividing 15 KRAS strands by A CTB strands and multiplying by 100, All mutations reporting to the F AM dye are quantified using KRAS 38A calibrators, and all mutations reporting to the H TEX dye are quantified using KRAS 35C calibrators. The calibrators for all three dyes show similar linearity (Figure 4) and good reproducibility. The assay was optimized to niniize cross reactivity with KRAS WT plasmid at 200,000 strands per reaction which wias approximately 20 0.07 to 0.11 percent mutation in the 3-dye configuration. RESULTS The 2-dye KRAS multiplex QuARTS assay was evaluated using 87 tissue samples 25 consisting of 19 normal, 16 adenoma and 52 colorectal cancer samples. KRAS QuARTS results showed good agreement with sequencing data. All normal colon samples had a value equal to or less than 0,55 percent mutation. All of the sampls that were KRAS positive by sequencing were greater than 8 35 percent nutation. The colorectal cancer and adenoma samples that were KRAS negative by sequencing showed a range from 0.04 to 1.33 percent 30 mutation with a mean of 0,4 ± Q 29%,i ablels 3 and 4; shown in Fig. 6). The 3-dye KRAS multiplex QuARTS assay was evaluated using 191 tissue samples consisting of 47 normal, 48 adenoma, and 96 colorectal cancer samples (Tables 2 below and 'Tables 5 and 6 shown in Fig 6, fhis set included 86 of the 87 tissues that were also tested with the 2-dye configuration (Table 4; shown in fig 6). KRAS QuA RTS showed excellent 35 agreement with sequencing data, All of the samples that were KR AS positIve by sequencing 25 showed at least2,45 percent mutation in the KRAS QuARTS assay, with a mean of 373 ± 33 3 percent mutation. 'Ihe colorectal cancer and adenoma sampks that were negative by sequencing showed a range from 0.00 to 1,99 percent mutation with a mean of 0.14 ± 033 percent mutadon The adenoma sample that showed 1.99 percent mutation was detected at 5 1 33 percent mutation in the 2-dye configuratonAll normal colon samples had a value equal to or less than 0.21 percent mutation (mean for normal samples was 0.03 ± 0,04 percent mutation), Taole 2. Sequence and QuARTS assay data concordance: Subset of Tissue DNA Average ACTB l% mutation. Sample Colon tissu# % muttion strands- F AM Gntp am hHtoogEX channel 1 Cancer 11090 2A5% 104% 35A TS2 Adenoma 5187 11.74% 6 u30% 35A TS3 Cancer 2180 19 29% 003% 35T TS4 Adenoma 1172 37 80% 0.02% 35T T15 Cancer 46798 55 69% 0.00% 38A TS6 Cancer 64399 3 46% 61,78% 34A TS7 Adenoma 3349 16 16% 19.20% 35A TS8 Cancer 7578 12 60% 0.00% 34T T59 Adenoma 18667 199% 0.07% WT T510 Cancer 52344 0.06% 000% WT TS11 Cancer 20977 0,02% 0,00% WT TS12 Adenoma 51648 001% 0.52% WT TS13 Adenoma 43957 0.03% 0,01% WT T514 Adenoma 98260 0 12% 0.30% WT TS15 Normal colon 21484 0 00% 0-00% WT TS16 Normal colon 92492 0 03% 000% WT T517 Norrnal colon 13228 0 01% 0.00% WT TS18 Normal colon 579 0;00% 0.00% WT S119 Normal color 211699 0.00% 0.18% W TS20 Normal colon 85889 0.01% 0.02% WT T521 Adenoma 4097 63,83% 4134% 34T 350 TS22 Cancer 1806 101,96% 0.00% 38A 1523 Adenorna 1864 1.37% 43,58% 34A TS24 Adenoma 6433 48.83% 4,70% 35T 10 The KRAS QuARTS multiplex assay showed a maximum of 0 11 percent mutation forcossWeacdivity with KRAS WT plasmid control at 200,000 strands per reaction. 26 Figure 5 shows the distribution of percent rmutation by sample type. With the highest normal giving 0.21 percent and the lowest sequencing confirmed KRAS mutation at 2.A5 percent the assay agrees 100% on those samples Because of the higher sensitivity of the QuARTS assay 2 cancers and 12 adenomas are observed that are elevated above the highest 5 percent mutation of the normal samples Based on sequencing data: the 52 CRC samples contained 22 KRAS mutations and 30 wild-type genotypes, the 16 adenomas 1cm contained 8 mutations and 8 wild-type genotypes, and the 19 normal tissues contained all wild-type genotypes. The QuARTS assay detected 100% of the KRAS mutations in the CRC and adenomas and provided excellent 10 differentiation between wild-type and mutation, with the highest percent KR AS mutation of nomal wild-type samples at 055% and the lowest percent mutation of KRAS positive samples at 8.34%. Based on this data. this assay is more sensitive analyically than standard sequencing. In this study we were able to show results for 6 of the 7 mutations detected by the 1S assay; mutation 34C (Gly12Arg) in exon 2 represents 0.5 % of KRAS mutations in colorectal cancers and was not represented in these samples. Using plasmid derived sequences we have shown the assay is capable of detecing this mutation (data not shown) This multiplex does not distinguish among mutations. The assay shows some cross reactivity between mutations which is likely to improve sensitivity since the signal is 20 increased without any increase in WT cross reactivity, The three-dye configuration of the KRAS QuART'S multiplex assay showed better specificity than the 2-dye version when all KRAS mutations are reporting to a single dye, the signal from cross-reactivity with WT is additive but by distributing the KRAS mutation signal across two dyes, the cross-reactivity with WT is reduced by more than half, 25 EXAMPLE 2 MATERIALS AND METHODS Fig. 7 shows the designs used for multiplex detection and quantification of the seven mutant alleles of KRAS and the ACTB (beta actin) internal control. Three. 5-flaps (AS and 30 A7 for KRAS and Al for ACT1 1 were used in the assay. The probes with flaps A5 and A7, used for KRAS mutants, were med in conjunction with two FRET oligonuckoides A5 HEX and A7-FAM thus giving signal in these two dye channels for KRAS mutations. The ACTB probe- on the other hand, had a 5-flap Al-Quasar, resulting in Quasar 670 signal when ACTB is present. Further details of the reagent mix are set forth below. 27 Reagent mix comoonents Mutation QuARTS Assay Primers Conc infinal pMer Name Sequence KRAS RPIO GATTCTGA2ATTCTTATCGT 1SEQ ID NO: 5) 350 KRAS 0A P2C ACTTGTGGTAGTGAGTCA (SEQ ID ND: 1 250 kRASsTP20 ACTGTnA GAGeTcT (SEQ ID NO: 2) 250 KRAS35CP4A AACTITGSTAGTTCAATC (SEQ ID NO' W 250 KRAS 34A P2C 19b C GTGGTAGTTGGAG0A (SEQ ID NO 4 250 KRAS 4TP2C TGTTAGTTGGAGCCT (SEQ ID No' 5) 250 TTATTGTFGGTAG--T-- GAC-CTC KRAS 34C P4Cb

T

A

T

? 'GAC-TC (SEQ ID N: 6) 250 KRAS A P2A 19b TGGTAGTTOGAGCTGGTAA (SEQ ID NO: 7) 250 ACTBWTFP3 CCATNAGCTGGTGTAAAG (SEQ ID NO: 9 " 150 CTWCTGTGCACCTACTTAATACAC (SEQ ID NO : 150 Mutation QuARTS Assay Probes Pbes .Conehirtfinal ------ c-on- (-M) AlAS 31 ' Pb GCOCGTCIOT T GG AU GCA /326/ f:m ( SEQ IDN31 K RAS 35> AS Pb ACsccm-coT'3/ -. Az ;Rb{ (SQ ID NO: 12 310 CACGGACGATGGCGTAGGCA/ 3:/ __ __ _ __ _ __ __ _ __ _ __ SEQ ID NO' M' 3 110 ----- -- --- -- --- -- --- -- --- -- --- --- 2L - KRa35A A5 bP] CCGMTCTGAW USEQ 1D NO: 13 310 KRA .S 38A A JIi P b W,,C7CCTAM M-

------

(SEQ ID NO' 14) 310 GCCGTCCTGTSCGTAGGC/3C6 (SEQ ID NO' 15) 310 CANCGGACGCGTGGCGTAG350/3C8 KRAS 34C A5 Pb CSEQ ID Nw jn) 310 S Q ID NO; 16 310 CCACGGACGAGTGCTAGGC KRAS 34A A5 pb (SQ ID NO: 17) 310 A 'BWT b Al NTVo 3 __(SEQ TD ND: 10R)31 FRET cassettes al FRET sequentes exactly mat ch the arm landing pad (no extra 3 bases) Con cin fi7 name equence5--reac--- Arm 5 TAMRA FRET K(SEQ ID No 19 100 Arm 7 FAM FRET {SEQ ID NO: 20) .100 Arm 1 Quasar 670 FRET SEQ ID NO: 2 100 2S Other components Current reaction buffer componett._____________ Reagents C..oncentration per reaction recombinant HotStart Go Taq 0 07U/uL water, PM1009 NA PM1143, Eluion Buffer Te ova Te pH 8.0 buffer + 2Ong/uL tRNA dNTPs* 250pM MOPS 10mM IM KCI OJ97mM 2M MgCI2 75mM 5M Tris-HCI pH 8 0.319mM 50% Tween-20 0,08% 20% GEPAL 0308% 80% glycerol 25% Cleavase 73 ng/uL BSA 1 (3ng/uL (3ug/ 30DuLreaction) likely to be part of oligo mix rather than 20X reaction buffer Theriocvcinge Parameters 95*C /20' 100% Amplification 1 4c/3 100%1 7*/30" 100% 95"/ 20"10 Ampilication 2 530C /1 100 35 700c / 30 100% coonng40 /3 100%1 RESULTS Using an oligormeleotide miixture with three FRET oligonucleotides (FAMI or HEX with Quasar) and one, KRAS nutant and ACTB specific oligon-ucleotide mixture, it was 10 found that. Signal is, generated for ACTB only when A CTBR is present. However; for KR AS, it was found that cross-reactive signal is ge~nerated in HEX and FAN/M chianneol between some mutant KRAS muLtatians (see table belw) wvith miinimail cross -reactivity sipnal for wild-type KRAS For example. when thea is KRAS 34C mutation the 35A and 35T Amplificaiew tions 1ac ap/p 10%1 Using an oligonucleotide mitable crossreactivE' signal The table below shows the 15 resuas reported as cycle threshold values obtained using 000 copies of the different KRAS mutant and 10a000 copies for ACT targets with eiier duplex or tip sigonucleotide 29 mixes, Based on these results, and based on the low cross reactivity of 35C and 38A targets, those two targets were selected to be the calibrators used for standard curve generation. Tarar 'A 4C 34T 35 35C 352 dSA 36 I AB 14 NYC Rcoororg Ovs 0bs mix 1 2 4 S 7 16 9 10 NEX/OuesAr 34A 2 3A66 335 3l) 1RS 281 NEX/uasar 34C B 1 13 ./ 224 31 6 3.8 26.4 EA W/QUQS 3tf C 225 202 106 26.S 34.3 28, 9.2 214 HEtuxA 5 A 25 136 24 S 29 23.4 27,9 11 14 26. W<X/Qaer 9SC E 29,4 305 199 1> 162 299 104 262 'AM/Qunar 30 F 272 14l 253 220 2 2.6 6.6 26.5 FA/usr 36A [x G >1 3267 6 320T 23.5____ 134.494 20 26.0 FAM/EX/u at Ad MW ipke H 1 1 41 12 C /18 6 15/, 6 24 2/ -12,71 363 961 542 5 When a plasmid containing a triple insert of 2 KRAS mutations (35C & 38A) and one ACTB (i.e., 35C/38A/ACTB plasmlid was used as calibrators (i.e. standard curves) to calculate strand numbers for all other stations data showing the following was obtained: a) Less than 0.05% t cross reactivity between the multiplex KRAS mutant 0 oligonucieoide mixes and wild-type KRAS. b) 35C standard curve calibrator can be used to quantify the HEX-reporting mutants (35A, 35C, 34A, 34C) and, siilarly, iat the 38A standard curve calibrator can be used to quantify the FAM-reporting mutants (38A, 34T, 35T). c) The assay can be used for both detecton (ie. screening for mutations) and 15 quantification of KRAS nmutants with minimal cross-reactivity with wiid-type. d) The sensitivity of the assay is approximately a single-copy per reaction. Additionally, using the multiplex KRAS mutant assay designs for screening previously sequenced tissue samples by assigning the HEX signal 135C calibrator) as an 20 indicator for the presence of the 35A, 35 34A,34C mutations and the FAM signal (38A calibrator) as an indicator for the presence of the 38A, 34T, 35T mutations the following results were obtained: Tes t of tSsue and stool s:ampes wit3 C oIpr iKAS QAA RTS s Geotypeby C.titsue K RAS3csc IRA38A' ACTB t m4ds ACiW$It 'nds m e--en-in5 b .s td .- y -trnds- I . .. nds . 5C- -A) - - --... Tissue Samples 1 T

---_---

0SaDA G34T ADENMA - 385 1,998 22,346E 0 30% Poitv 036DA A G3A CANCER 641 _06 1_ 4.890 5,953 11% 18% Posive 029DAA G34A CANCER 34833 2,509 6382 72,416 62% 3% Positive 089DAA G05A ADENOMA 126 180 1821 2,098 7% 9% Positive 056DAA G35T ADENOMA 0 48. LOG3 1,282 0% 38% Positive 026DAA G38A CANCER - 29,200 M,166 523 5- SG Positive 0190AA WT normal colon -42,028 51,976 0% 0% Ngtisve 0080AA WVT normal colon IS- 3 [ -39,535 48,897 0% 0% Negative 092 DA A WT normal colon -3- 6 [ SL1.0S 100,809 0% 0% NegatIve 017DAA WT normal colon 32 113.608 149,212 0% 0% Nea tive 013D3:AA WT normal colon S 50 32 99,401 130,064 T0% 0% Negative 51 G35T ADENOMA 33 5,353 18.647 22,351 0% 24% Positive 517 G38A CANCER 5,227 9,971 :11,816 0% 44% Positive 540 GSdT CANCER 14,732 456 16,462 0% 85% Positive S4 WNT normal colon - 6 36,6211 0% 0% Negative 585 WVT normal colon 1 | 11.982 14,111 0% 0% Negative 586 WT normalcolot - 63 161567 206,770 0% 0% Negative 87T normal colon 22,006 26,459 0% 0% Noetive Gno_ _y__ Cl tosu 35C K_3 ASta n ACME Seg::.. g hIdy St$md sthands ( .. C :SA uttir uaic Cl IEXPA 13.2 38G>A CANCER 4 15,845 4?028 36,827 0% 33% Positive 511 34G>T CANCER 22473 I 1072 SA1 0% 23% Posi ve S8 >SG>A CANCER 1.156 379 129,130 1i 1% Positive S935G>T ADENOMA 122 17 057 59,046 45 326 Ok 29% Positive S2 3sG CANCER 44 . ,7 72221 55,335 0% 1% Positive 7 3l 7 G > A CANCER 1 11,44] 117,166 88,406 0% 10% Positive 35G>T CANCUR 2 956 12.573 9,921 0?8% Positive 235GnT ArENOA 20 474 27,373 21,292 0% 2 Positve Two plasmids, SC/ACTB and 38A/ACTS, were used for generation of standard curves for HEX/Quasar and FAM/Quasar, respectively. 5 ** ACT Strands (3c) are calculated based on the ACT/Quasar standard curve generated using the 35c/ACT plasmid, * ACTE Strands (38A are calculated basedon the ACTB/Quasar standard curve generated using the 38A/ACTB plasmir. The data shows full agreement hetween QuARTS and sequencing. This indicates that the KRAS mutant QuARTS assay can be used on both stool DNA as well as dssue samples to screen for KRAS mtatiOns. 31

Claims (12)

1. A reagent mixture comprising: a) amplification reagents comprising a thermostable polymerase, nucleotides, a set of at least seven forward primers, and a reverse primer, wherein: i. the 3' terminal nucleotide of each forward primer of said set base pairs with a different point mutation in the KRAS gene relative to other forward primers in said set, wherein said point mutaton is selected from the following point mutations: 34A, 10 34C, 34T, 35A, 35C, 351 and 38A; ii. each of said forward primers comprises a nucleotide sequence that is fully complementary to a sequence in said KR AS gene with the exception of a single base mismatch within 6 bases of said 3' terminal nucleotide: and iii. each of said forward pnmers, in combination with said reverse prmer 15 selectively amplifies a different allele of a KRAS gene, wherein the allele that is amplified is defined by the point mutation to which said 3' terminal nucleotide base pairs; and b) flap assay reagents comprising a flap endonuclease, a first FRET cassette that produces a fluorescent signal when cleaved, said set of at least seven forward primers, and a 20 corresponding set of at least seven different flap oligonucleotides that each comprise a ncleotide that base pairs with one of said point mutations; wherein said reagent mixture is characterized in that, when said reagent mixture combined with a nucleic acid sample that comprises at least a 1,00-fold excess of wild type copies of said KR/AS gene relative to mutant copies of said KRAS' gene that contain one of 25 said point mutations and thermocycled, said reagent mixture can amplify and detect the presence of said mutant copies of the KRAS gene in said sample.

2. The reagent mixture of claim 1, wherein said flap probe base pairs with 10 to 14 contiuuous nucleotides of said KRAS gene. 30

3. The reagent mixture of claim I, wherein the complementary nucleotide sequence of said forward primers is at least 16 nucleotides in length. 32 4, The reagent mixture of claim l, wherein said mismatch in said forward primers is, independently, at position -, posiion -2, position -3 position -4 or position -5 relative to said terminal nucleotide. 5 5, The reagent mixture of claim 1, wherein said forward pr ners comprise the last 12 contiguous nucleotides of the forward primers shown in Fig. 7.

6. The reagent mixture of claim 1, further comprisinog a nuclei acid sample that comprises at least a 100-fold excess of wild type copies of said KRAS gene relative to mutant 10 KR AS gene that contain one of said point mutations

7. The reagent mixture of claim 6, wherein said sample is obtained from a human,

8. The reagent mixture of claim 7, wherein said sample is a stool sample. 15

9. The reagent mixture of claim 7, wherein said sample is a tissue or biopsy sample.

10. The reagent mixture of claim I, wherein said reaction mixture further comprises second amplification reagents and second flap reagents for amplifying and detecting a control 20 sequence that is in a gene that is not in KR AS, wherein said second flap reagents comprise a second PRIET cassette that produces a signal that is distinuishable from the signal of the first FRET cassette. 1 L The reagent mixture of claim 10, Wherein said gene is 3-actin. 25

12. The reagent mixture of claim I wherein said flap assay reagems comprise a first FR ET cassette and a second FRET cassette that produce distinguishable fluorescent signals when cleaved, and wherein at least one of said at least seven different flap oligonucleotides coinprises a flap sequence that hybridizes to said first FRET cassette and the remai-nder of 30 said at least seven different flap oligonucleotides hybridizes to said second Ee c assette. 13, A method of sample analysis comprising: a) subjecting a reaction mixture comprising i. the reagent mixture of claim I and ii a nucleic acid sample that comprises at least a 100-fold excess of wild type copies of said 33 KRAS gene relative to mutant KRAS gene that contain one of said point mutations, to the following thermocycling conditions: a first st of 5- 15 cycles of: f.a first temperature of at least 90 "C; 5 ii. a second temperature in the range of 60 C to 75 "C; iii. a third temperature in the range of 65 "C to 75 "C; followed by: a second set of 20-50 cycles of: i. a fourth temperature of at least 90 'C; ii. a fifth temperature that is at least 10 C lower than said second temperature; 10 iii. a sixth temperature in the range of 65 "C to 75 "C: wherein no additional reagems are added to said reaction between said first and second sets of cycles and, in each cycle of said second set of cycles, cleavage of a flap probe is measured; and b) detecting the presence of a mutant copy of KRAS in said nucleic acid sample. 15 14, The method of clain 13, wherein said amplifying and detecting steps are done using a reaction mixture that contains both said PCR reagents and said flap reagents, and no additional reagents are added to said reaction mixture between said amplifying and detecting steps. 20

15. The method of clanm 13, wherein said nucleic acid sample comprises at least a ,00t> fold excess of wild type copies of said KRAS gene relative to mutant KRAS gene that contain one of said point mutations. 25 16. 'The method of claim 13, wherein said sample is obtained from a human.

17. The method of claim 13, wherein sample is stool.

183. The method of claim , further comprising making a diagnosis of colon cancer or 30 adenoma based on whether mutant copies of said KRAS gene are idenified in said stool 19. The method of clan 13, wherein said method comprises measuring cleavage of said flap probe whie said reaction mixture is at said fifth temperature. 34 20, The method of claim 13, wherein cleavage of said flap probe is measured by detecting fluorescence of said reaction mixture during each of said 20-50 cycles. 21 The method of claim 13, wherein said fourth temperature is in the range 50 C to 5 55 C 2 The method of claim 13, further comprising normalizing the amount of said mutant copy of KRAS in said nucleic acid sample relative to the amount of a control nucleic acid present in said sample thereby determining the amount of said mutant copies mutant copy of 10 KRAS in said sample, 23. A kit comprising: a) amplification reagents comprising a thermostabie polymerase, nucleotides, a set of at least seven forward primers, and a reverse. pmriMer, wherein: 1.5 i, the 3' terminal nucleotide of each forward primer of said set base pairs with a different point mutation in the KR AS gene relative to other forward primers in said set, wherein said point mutation is selected from the following point mutations: 34A-, 34C, 34T, 35A 35C, 35T and 38A; ii. each of said forward primers comprises a nucleotide sequence that is fully 20 complementary o a sequence in said KRAS gene with the exception of a single base mismatch within 6 bases of said 3' terminal nucleotide; and iii. each of said forward primers, in combination with said reverse primer, selectively amplifies a different allele of a KRAS gene, wherein the allele that is amplified is defined by the point mutation to which said 3' terminal nucleotide base 25 pairs; and b) flap assay reagents comprising a flap endonuclease, a FRET cassette, said set of at least seven forward primers, and a corresponding set of at least seven different flap oligonucleotides that each comprise a nucleotide that base pairs with one of said point 30 35