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 suspected of comprising at least a second allelic variant of the target sequence, 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 probe that is complementary to a region of the target sequence comprising the second allelic variant, wherein said region encompasses a position corresponding to the binding position of the allele-specific nucleotide portion of the first allele-specific primer, and wherein the first allele-specific blacker 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 the 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 first allelic variant of the target gene in the nucleic acid sample.   
     
     
         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:
 i) the nucleic acid sample; 
 ii) 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; 
 iii) a second allele-specific blocker probe that 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, 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 the 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 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, second or first and second allele-specific primer and/or said first, second, or first and second allele-specific blocker probe comprises at least one modified base. 
     
     
         6 . The method of  claim 5 , 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). 
     
     
         7 . The method of  claim 5 , wherein said modified base is a locked nucleic acid (LNA) base. 
     
     
         8 . The method of  claim 5 , wherein said modified base is a fdU or iso dC base. 
     
     
         9 . The method of  claim 5 , wherein said modified base is any modified base that increases the Tm between matched and mismatched target sequences or nucleotides. 
     
     
         10 . The method of  claim 5 , wherein said modified base is located at (a) the 3′-end, (b) the 5′-end, (e) at an internal position or at any combination of (a), (b) or (c) within said allele-specific primer and/or allele-specific blocker probe. 
     
     
         11 . The method of  claim 5 , wherein the specificity of said detecting is improved by the inclusion of said modified base in said first, second or first and second allele-specific primer and/or said first, second, or first and second allele-specific blocker probe as compared to when it is not. 
     
     
         12 . The method of  claim 11 , wherein said improvement is at least 2 fold. 
     
     
         13 . The method of  claim 1  or  3 , wherein the specificity of said detecting is improved by at least 2 fold as compared to the specificity of detecting an allelic variant in a nucleic acid sample using ASB-PCR methods. 
     
     
         14 . The method of  claim 1  or  3 , wherein said carrying out an amplification reaction comprises a 2-stage cycling protocol. 
     
     
         15 . The method of  claim 14 , wherein the number of cycles in the first stage of said 2-stage cycling protocol comprises fewer cycles than the number of cycles used in the second stage. 
     
     
         16 . The method of  claim 14 , wherein said number of cycles in the first stage is about 90% fewer cycles than said number of cycles in the second stage. 
     
     
         17 . The method of  claim 14 , wherein said number of cycles in the first stage is between 3-7 cycles and said number of cycles in the second stage is between 42-48 cycles. 
     
     
         18 . The method of  claim 14 , wherein the annealing/extension temperature used during the first cycling stage of said 2-stage cycling protocol is between 1-3° C. lower than the annealing/extension temperature used during the second stage. 
     
     
         19 . The method of  claim 14 , wherein said annealing/extension temperature used during the first cycling stage of said 2-stage cycling protocol is between 56-59° C. and said annealing/extension temperature used during said second stage is between 60-62° C. 
     
     
         20 . The method of  claim 1 , wherein said step (a) is preceded by a pre-amplification step. 
     
     
         21 . The method of  claim 20 , wherein said 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. 
     
     
         22 . The method of  claim 21 , wherein the products of said multiplex amplification reaction are divided into secondary single-plex amplification reactions, wherein each single-plex amplification reaction contains at least one primer set previously used in said multiplex reaction. 
     
     
         23 . The method of  claim 21 , wherein said multiplex amplification reaction further comprises a plurality of allele-specific blocker probes. 
     
     
         24 . The method of  claim 21 , wherein said multiplex amplification reaction is carried out for a number of cycles suitable to keep the reaction within the linear phase of amplification. 
     
     
         25 . A reaction mixture comprising:
 a) a nucleic acid molecule;   b) an allele-specific primer, wherein an allele-specific nucleotide portion of the allele-specific primer is complementary to a first allelic variant of a target sequence;   c) an allele-specific blocker probe that is complementary to a region of the target sequence comprising a second allelic variant, wherein said region encompasses a position corresponding to the binding position of the allele-specific nucleotide portion of the allele-specific primer, and wherein the allele-specific blocker probe comprises a minor groove binder;   d) a 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   e) a detector probe.   
     
     
         26 . The reaction mixture of  claim 25 , wherein said allele-specific primer and/or said allele-specific blocker probe comprises at least one modified base. 
     
     
         27 . The reaction mixture of  claim 26 , 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). 
     
     
         28 . The reaction mixture of  claim 26 , wherein said modified base is a locked nucleic acid (LNA) base. 
     
     
         29 . The reaction mixture of  claim 26 , wherein said modified base is a fdU or iso dC base. 
     
     
         30 . The reaction mixture of  claim 26 , wherein said modified base is any modified base that increases the Tm between matched and mismatched target sequences or nucleotides. 
     
     
         31 . The reaction mixture of  claim 26 , 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 said allele-specific primer and/or allele-specific blacker probe. 
     
     
         32 . A composition comprising:
 a) 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 a target sequence;   b) a first allele-specific blocker probe that is complementary to a region of the target sequence comprising the second allelic variant, wherein said region encompasses a position corresponding to the 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;   
     
     
         33 . The composition of  claim 32 , further comprising a 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. 
     
     
         34 . The composition of  claim 32  or  33 , wherein said first allele-specific primer and/or said first allele-specific blocker probe comprises at least one modified base. 
     
     
         35 . The composition of  claim 34 , 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). 
     
     
         36 . The composition of  claim 34 , wherein said modified base is a locked nucleic acid (LNA) base. 
     
     
         37 . The composition of  claim 34 , wherein said modified base is a fdU or iso dC base. 
     
     
         38 . The composition of  claim 34 , wherein said modified base is any modified base that increases the Tin between matched and mismatched target sequences or nucleotides. 
     
     
         39 . The composition of  claim 34 , 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 said allele-specific primer and/or allele-specific blocker probe. 
     
     
         40 . A kit comprising, two or more containers comprising the following components independently distributed in one of the two or more containers:
 a) 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 a target sequence; and   b) a first allele-specific blocker probe that is complementary to a region of the target sequence comprising the second allelic variant, wherein said region encompasses a position corresponding to the 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.   
     
     
         41 . The kit of  claim 40 , further comprising a 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. 
     
     
         42 . The kit of  claim 41 , wherein said first allele-specific primer and/or first allele-specific blocker probe comprises at least one modified base. 
     
     
         43 . The kit of  claim 42 , 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). 
     
     
         44 . The kit of  claim 42 , wherein said modified base is a locked nucleic acid (LNA) base. 
     
     
         45 . The kit of  claim 42 , wherein said modified base is any modified base that increases the Tin between matched and mismatched target sequences and/or nucleotides. 
     
     
         46 . The kit of  claim 42 , wherein said allele-specific blocker probe comprises an MGB moiety at the 3′-end, the 5′-end and/or at an internal position within said allele-specific blocker probe. 
     
     
         47 . A method for detecting a first allelic variant in a target sequence in a nucleic acid sample, comprising:
 a) forming a reaction mixture comprising:
 i) a nucleic acid sample; 
 ii) an allele-specific primer, wherein an allele-specific nucleotide portion of the allele-specific primer is complementary to the first allelic variant of the target sequence; 
 iii) an allele-specific blocker probe that is complementary to a region of the target sequence comprising a second allelic variant, wherein said region encompasses a position corresponding to the binding position of the allele-specific nucleotide portion of the allele-specific primer, and wherein the allele-specific blocker probe comprises a blocking moiety; 
 iv) a 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 detector probe; 
   b) PCR amplifying the target sequence using a 2-stage cycling protocol, comprising:
 i) a first amplification step comprising a first number of cycles run at a first annealing/extension temperature; and 
 ii) a second amplification step comprising a second number of cycles run at a second annealing/extension temperature, 
   wherein the first number of cycles is fewer than the second number of cycles and the first annealing/extension temperature is lower than said second annealing/extension temperature.   c) detecting a change in a detectable property of the detector probe in the amplified products of the target sequence produced by step (b), thereby detecting the first allelic variant of the target gene in the nucleic acid sample.   
     
     
         48 . The method of  claim 47 , wherein said number of cycles in the first step is about 90% fewer cycles than said number of cycles in the second step. 
     
     
         49 . The method of  claim 14 , wherein said number of cycles in the first step is between 3-7 cycles and said number of cycles in the second step is between 42-48 cycles. 
     
     
         50 . The method of  claim 14 , wherein the annealing/extension temperature used during the first step of said 2-stage cycling protocol is between 1-3° C. lower than the annealing/extension temperature used during the second step. 
     
     
         51 . The method of  claim 14 , wherein said annealing/extension temperature used during the first step of said 2-stage cycling protocol is between 56-59° C. and said annealing/extension temperature used during said second step is between 60-62° C. 
     
     
         52 . A method for detecting an allelic variant of a target sequence in a nucleic acid sample, comprising:
 a) forming in a single vessel a first reaction mixture comprising:
 i) a nucleic acid sample; and 
 ii) at least two sets of primers wherein each set comprises (a) a first allele-specific primer and a second allele-specific primer, wherein the allele-specific nucleotide portion of said first and second allele-specific primers is complementary to a first allele and a second allele, respectively, of a given SNP within a target sequence; and (b) a locus-specific primer that is complementary to a region of said target sequence that is 3′ from the first and second alleles and on the opposite strand; wherein the at least two sets of primers is each specific for a different target sequence; 
   b) amplifying said different target sequences using a number of cycles suitable to keep the reaction within a linear phase;   c) dividing the amplified products of step (b) into at least two separate vessels;   d) adding to each of the divided products of step (c)
 i) at least one set of primers used in step (a), 
 ii) an allele-specific blocker probe that is complementary to a region of said target sequence comprising a second allelic variant, wherein said region encompasses a position corresponding to the binding position of the allele-specific nucleotide portion of the first allele-specific primer, and wherein the first allele-specific blacker probe comprises a minor groove binder; and 
 iv) a detector probe 
 to form a second and a third reaction mixture in the at least two separate vessels; 
   e) amplifying said target sequence in said second and third reaction mixtures; and   f) detecting a change in a detectable property of the detector probe in each of the amplified products of said target sequence produced by step (e), thereby detecting the allelic variant of the target gene in the nucleic acid sample.   
     
     
         53 . The method of  claim 52 , wherein said first reaction mixture is a multiplex reaction and said second and third reaction mixtures are single-plea reactions.