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 at least one single nucleotide polymorphism (SNP) in a first allelic variant and a second allelic variant of a target sequence in a nucleic acid sample, the method comprising:
 a) forming a first reaction mixture by combining:
 i. the nucleic acid sample that contains the first allelic variant of the target sequence; 
 ii. a first allele-specific primer, wherein an allele-specific nucleotide portion of the first allele-specific primer is specific to the at least one SNP present in the first allelic variant of the target sequence; 
 iii. a first allele-specific blocker probe that is complementary to a region of the target sequence on the second allelic variant, wherein said region encompasses a position corresponding to a binding position of the allele specific nucleotide portion of the first allele-specific primer, and wherein the first allele-specific blocker probe comprises a minor groove binder; 
 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 an opposite strand; and 
 v. a first detector probe; 
   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 detector probe, thereby detecting the at least one (SNP) in the first allelic variant of the target sequence in the nucleic acid sample.   
     
     
         2 . The method of  claim 1 , wherein the at least one SNP is an A to G SNP, a C to T SNP, a C to G SNP or an A to T SNP. 
     
     
         3 . The method of  claim 1 , wherein the at least one SNP is a nucleotide insertion or a nucleotide deletion. 
     
     
         4 . The method of  claim 1 , wherein the allele-specific nucleotide portion of the first allele-specific primer has at a 3′ allele-specific nucleotide portion an A or G to aid in detecting an A to T SNP or a C to G SNP. 
     
     
         5 . The method of  claim 1 , further comprising:
 d) forming a second reaction mixture by combining:
 i) the nucleic acid sample; 
 ii) a second allele-specific primer, wherein an allele-specific nucleotide portion of the second allele-specific primer is specific to the at least one SNP present in the second allelic variant of the target sequence; 
 iii) a second allele-specific blocker probe that is complementary to a region of the target sequence on the first allelic variant, wherein said region encompasses a position corresponding to a binding position of the allele-specific nucleotide portion of the second allele-specific primer, and wherein the second allele-specific blocker probe comprises a minor groove binder; 
 iv) 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 an opposite strand; and 
 v) a second detector probe; 
   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 detector probe, thereby detecting the at least one (SNP) in the second allelic variant of the target sequence in the nucleic acid sample.   
     
     
         6 . The method of  claim 5 , wherein the at least one SNP is an A to G SNP, a C to T SNP, a C to G SNP or an A to T SNP. 
     
     
         7 . The method of  claim 5 , wherein the at least one SNP is a nucleotide insertion or a nucleotide deletion. 
     
     
         8 . The method of  claim 5 , wherein the allele-specific nucleotide portion of the second allele-specific primer has at a 3′ allele-specific nucleotide portion an A or G to aid in detecting an A to T SNP or a C to G SNP. 
     
     
         9 . The method of  claim 1 , further comprising using the change in the detectable property of the first detector probe to quantitate the first allelic variant possessing the at least one SNP. 
     
     
         10 . The method of  claim 5 , further comprising comparing the change in the detectable property of the first detector probe in the first reaction mixture to the change in the detectable property of the second detector probe in the second reaction mixture. 
     
     
         11 . The method of  claim 5 , wherein at least one of the first allele-specific primer, the second allele-specific primer, the first allele-specific blocker probe or the second allele-specific blocker probe comprises at least one modified base. 
     
     
         12 . The method of  claim 11 , wherein said modified base is an 8-aza-7-deaza-dN (ppN) base analog, where N is adenine (A), cytosine (C), guanine (G), or thymine (T). 
     
     
         13 . The method of  claim 11 , wherein said modified base is located at (a) the 3′-end, (b) the 5′-end, (c) at an internal position or at any combination of (a), (b) or (c) within at least one of the first allele-specific primer, the second allele-specific primer, the first allele-specific blocker probe or the second allele-specific blocker probe. 
     
     
         14 . The method of  claim 5 , wherein step (b) and/or step (e) comprise a 2-stage cycling protocol. 
     
     
         15 . The method of  claim 14 , wherein the number of cycles in a first stage of said 2-stage cycling protocol comprises fewer cycles than the number of cycles used in a second stage. 
     
     
         16 . The method of  claim 14 , wherein a first annealing/extension temperature used during the first cycling stage of said 2-stage cycling protocol is between 1-3° C. lower than a second annealing/extension temperature used during the second stage. 
     
     
         17 . The method of  claim 5 , wherein step (a) is preceded by a pre-amplification step. 
     
     
         18 . The method of  claim 17 , wherein the pre-amplification step comprises a multiplex amplification reaction that uses at least two complete sets of allele-specific primers and locus-specific primers, wherein each set is suitable or operative for amplifying a specific polynucleotide of interest. 
     
     
         19 . The method of  claim 18 , wherein said multiplex amplification reaction further comprises a plurality of allele-specific blocker probes. 
     
     
         20 . The method of  claim 18 , wherein said multiplex amplification reaction is carried out for a number of cycles suitable to keep the reaction within the linear phase of amplification.