Methods for variant detection

A method of detecting one or more variations in a target DNA sequence, the method comprising:

• (a) providing a first reaction mixture comprising:
• (i) a first allele-specific oligonucleotide primer and a second allele-specific oligonucleotide primer, both having a cleavage domain positioned 5′ of a blocking group and 3′ of a position of variation, the blocking group linked at or near the end of the 3′-end of the oligonucleotide primer, wherein the blocking group prevents primer extension and/or inhibits the first and second allele-specific oligonucleotide primers from serving as a template for DNA synthesis,
• (ii) a nucleic acid sample that may or may not have the target DNA sequence, wherein the target DNA sequence may or may not have the variation,
• (iii) a cleaving enzyme, and
• (iv) a polymerase, wherein the polymerase is a high-discrimination mutant H784Q Taq polymerase;
• (b) hybridizing the first and second allele-specific oligonucleotide primers to the target DNA sequence, if present in the sample, to form a double-stranded substrate;
• (c) cleaving the first and second allele-specific oligonucleotide primers hybridized to the target DNA sequence, if the first and second allele-specific oligonucleotide primers are complementary at the variation, with the cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the first and second allele-specific oligonucleotide primers;
• and
• (d) extending the first and second allele-specific oligonucleotide primers with the high-discrimination mutant H784Q Taq polymerase;
• and
• (e) providing a second reaction mixture comprising:
• (i) the first allele-specific oligonucleotide primer and a third allele-specific oligonucleotide primer, both having a cleavage domain positioned 5′ of a blocking group and 3′ of a position of variation, the blocking group linked at or near the end of the 3′-end of the first and third allele-specific oligonucleotide primers, wherein the blocking group prevents primer extension and/or inhibits the first and third allele-specific oligonucleotide primers from serving as a template for DNA synthesis,
• (ii) a nucleic acid sample that may or may not have the target DNA sequence, wherein the target DNA sequence may or may not have the variation,
• (iii) a cleaving enzyme, and
• (iv) a polymerase, wherein the polymerase is a high-discrimination mutant H784Q Taq polymerase;
• (f) hybridizing the first and third allele-specific oligonucleotide primers to the target DNA sequence, if present in the sample, to form a double-stranded substrate;
• (g) cleaving the first and third allele-specific oligonucleotide primers hybridized to the target DNA sequence, if the first and third allele-specific oligonucleotide primers are complementary at the variation, with the cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the first and third allele-specific oligonucleotide primers;
• and
• (h) extending the first and third allele-specific oligonucleotide primers with the high-discrimination mutant H784Q Taq polymerase, and detecting primer extension products;
• wherein the first, second, and third allele-specific oligonucleotide primers contain a 5′ tail sequence that comprises a universal primer sequence and a first, a second, or a third reporter probe sequence that corresponds to the first, the second, or the third allele-specific oligonucleotide primer, respectively, wherein the 5′ tail sequence is non-complementary to the target DNA sequence;
• and wherein the method is performed using real-time PCR.