Methods, compositions, and kits for detecting allelic variants

Abstract

In some embodiments, the present inventions relates generally to compositions, methods and kits for use in discriminating sequence variation between different alleles. More specifically, in some embodiments, the present invention provides for compositions, methods and kits for quantitating rare (e.g., mutant) allelic variants, such as SNPs, or nucleotide (NT) insertions or deletions, in samples comprising abundant (e.g., wild type) allelic variants with high specificity and selectivity. In particular, in some embodiments, the invention relates to a highly selective method for mutation detection referred to as competitive allele-specific TaqMan PCR (“cast-PCR”).

1 . A method for detecting a first allelic variant of a target sequence in a nucleic acid sample, comprising: 
       a) forming a first reaction mixture by combining:
 i. the nucleic acid sample; 
 ii. a first allele-specific primer, wherein an allele-specific nucleotide portion of the first allele-specific primer is complementary to the first allelic variant of the target sequence; 
 iii. a first allele-specific blocker that:
 a. is non-extendable at the 3′ end; 
 b. is complementary to a region of the target sequence comprising a second allelic variant of the target sequence, wherein said region encompasses a position corresponding to the binding position of the allele-specific nucleotide portion of the first allele-specific primer; 
 c. does not comprise a label; and 
 d. comprises a minor groove binder located at the 3′-end, the 5′-end and/or at an internal position within said first allele-specific blocker and; 
 
 iv. a first locus-specific primer that is complementary to a region of the target sequence that is 3′ from the first allelic variant and on the opposite strand; and 
 v. a first locus-specific detector probe having at least one label; 
 
       b) carrying out an amplification reaction on the first reaction mixture using the first locus specific primer and the first allele-specific primer to form a first amplicon; and 
       c) detecting the first amplicon by detecting a change in a detectable property of the first locus-specific detector probe, thereby detecting the first allelic variant of the target gene in the nucleic acid sample at a sensitivity level of a single copy of the first allelic variant in a background of about 1×10 5  to about 1×10 6  copies of the second allelic variant. 
     
     
         2 . The method of  claim 1 , further comprising using the change in a detectable property of the first detector probe to quantitate the first allelic variant. 
     
     
         3 . The method of  claim 1 , further comprising: 
       d) forming a second reaction mixture by combining:
 vi. the nucleic acid sample; 
 vii. a second allele-specific primer, wherein an allele-specific nucleotide portion of the second allele-specific primer is complementary to the second allelic variant of the target sequence; 
 viii. a second allele-specific blocker that:
 a. is non-extendable at the 3′ end; 
 b. is complementary to a region of the target sequence comprising the first allelic variant, wherein said region encompasses a position corresponding to the binding position of the allele-specific nucleotide portion of the second allele-specific primer; 
 c. does not comprise a label; and 
 d. comprises a minor groove binder located at the 3′-end, the 5′-end and/or at an internal position within said second allele-specific blocker and; 
 
 ix. a second locus-specific primer that is complementary to a region of the target sequence that is 3′ from the second allelic variant and on the opposite strand; and 
 x. a second locus-specific detector probe having at least one label; 
 
       e) carrying out an amplification reaction on the second reaction mixture using the second allele-specific primer and the locus-specific primer, to form a second amplicon; and 
       f) detecting the second amplicon by detecting a change in a detectable property of the second locus-specific detector probe, thereby detecting the second allelic variant of the target gene in the nucleic acid sample. 
     
     
         4 . The method of  claim 3 , further comprising comparing the change in a detectable property of the first detector probe in the first reaction mixture to the change in a detectable property of the second detector probe in the second reaction mixture. 
     
     
         5 . The method of  claim 1  or  3 , wherein said first and/or second allele-specific primer comprises a tail. 
     
     
         6 . The method of  claim 5 , wherein said tail is GC-rich. 
     
     
         7 . The method of  claim 5 , wherein said tail is between 2-30 nucleotides long. 
     
     
         8 . The method of  claim 5 , wherein said tail is at the 5′ end of said first and/or second allele-specific primer. 
     
     
         9 . The method of  claim 1  or  3 , wherein the Tm of said first and/or second allele-specific primer is between 50° C. to 55° C. 
     
     
         10 . The method of  claim 1  or  3 , wherein said concentration of said first and/or second allele-specific primer is between 20-900 nM. 
     
     
         11 . The method of  claim 1  or  3 , wherein said first and/or second allele-specific primer is designed to comprise a highly discriminating base at the 3′ terminus. 
     
     
         12 . The method of  claim 1  or  3 , wherein said allele-specific nucleotide portion of said first and/or second allele-specific primer is located at the 3′ terminus of said first and/or second allele-specific primer. 
     
     
         13 . The method of  claim 12 , wherein A or G is used as the 3′ allele-specific nucleotide portion of said first and/or second allele-specific primer if A/T is the allelic variant; or C or T is used as the 3′ allele-specific nucleotide portion of said first and/or second allele-specific primer if C/G is the allelic variant. 
     
     
         14 . The method of  claim 1  or  3 , wherein said first and/or second allele-specific primer and/or first and/or second allele-specific blocker comprises at least one modified base. 
     
     
         15 . The method of  claim 14 , wherein said modified base is a 8-aza-7-deaza-dN (ppN) base analog, where N is adenine (A), cytosine (C), guanine (G), or thymine (T). 
     
     
         16 . The method of  claim 14 , wherein said modified base is a locked nucleic acid (LNA) base. 
     
     
         17 . The method of  claim 14 , wherein said modified base is any modified base that increases the Tm between matched and mismatched target sequences and/or nucleotides. 
     
     
         18 . The method of  claim 14 , wherein the allele specific blocker comprises a non-extendable blocker moiety at the 3′ terminus. 
     
     
         19 . The method of  claim 18 , wherein the non-extendable blocker moiety is selected from an MGB, a modification of a ribose ring 3′-OH moiety of the allele specific blocker oligonucleotide, an amine (NH 2 ), a biotin, a PEG, a DPI 3 , and a PO 4 . 
     
     
         20 . The method of  claim 1 , wherein said MGB moiety or moieties is/are not cleaved from said first and/or second allele-specific blocker during said amplification reaction. 
     
     
         21 . The method of  claim 1  or  3 , wherein said first and/or second locus-specific detector probe has two labels. 
     
     
         22 . The method of  claim 21 , wherein said locus-specific detector probe is a 5′ nuclease probe. 
     
     
         23 . The method of  claim 1 , wherein said nucleic acid sample is genomic DNA (gDNA). 
     
     
         24 . The method of  claim 1  or  3 , wherein said first and/or second reaction mixture further comprises a polymerase, dNTPs, and/or other reagents or buffers suitable for PCR amplification. 
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 1  or  3 , wherein said amplification reaction is a real time polymerase chain reaction (PCR). 
     
     
         27 . The method of  claim 1 , wherein said nucleic acid sample is derived from a tumor sample, a blood sample comprising circulating tumor cells, a breast or a lung cancer tumor sample, a tumor comprises mutations in Ras, EGFR, Kit, pTEN, and/or p53, a tumor wherein a Ras mutation is a KRAS or and NRAS mutation, a tumor wherein a KRAS mutation is in codon 12 and/or codon 13 as depicted in  FIG. 6 . 
     
     
         28 - 91 . (canceled) 
     
     
         92 . The method of  claim 1  or  3  performed using a kit comprising: 
       two or more containers comprising the following components independently distributed in one of the two or more containers: 
       a) the first allele-specific primer; and 
       b) the first allele-specific blocker; 
       c) the locus-specific primer that is complementary to a region of said target sequence that is 3′ from said first allelic variant and on the opposite strand; and 
       d) a locus-specific detector probe wherein the detector probe is a 5′ nuclease probe. 
     
     
         93 .- 118 . (canceled) 
     
     
         119 . The method of  claim 1  or  3 , wherein said method has a selectivity of detection of at least 1:1000, 1:10,000, 1:100,000 or 1:1,000,000. 
     
     
         120 . The method of  claim 21 , wherein the two labels of said locus-specific detector probe comprise a fluorophore and a quencher. 
     
     
         121 . The method of  claim 120 , wherein said locus-specific detector probe further comprises a minor groove binder. 
     
     
         122 . The method of  claim 1  or  3 , wherein said first or second allele-specific blocker is at a concentration that is less than the concentration of said first or second allele-specific primer. 
     
     
         123 . The method of  claim 1  or  3 , wherein said second allelic variant is located 7-15 nucleotides away from said minor groove binder when said allele-specific blocker is hybridized to said target sequence. 
     
     
         124 . The method of  claim 1  or  3 , wherein said allele-specific blocker does not comprise a base analog. 
     
     
         125 . The method of  claim 1  or  3 , wherein said allele-specific primer and said allele-specific blocker both comprise one or more base analogs. 
     
     
         126 . The method of  claim 1  or  3 , wherein said allele-specific blocker has a Tm that is between 60° C. to 66° C. and said allele-specific primer has a Tm that is between 50° C. to 70° C.