Allele-specific amplification using a composition of overlapping non-allele-specific primer and allele-specific blocker oligonucleotides

An oligonucleotide composition comprising:

• (a) a first blocker oligonucleotide comprising a first sequence comprising a first target-neutral subsequence and a first blocker variable subsequence, wherein the first target-neutral subsequence comprises a first portion and a second portion, wherein the first blocker variable subsequence is flanked on its 3′ end by the first portion of the first target-neutral subsequence and on its 5′ end by the second portion of the first target-neutral subsequence, and is continuous with the first and second portions of the first target-neutral subsequence, wherein the first target-neutral subsequence is complementary to a first portion of a homologous subsequence of both a first target nucleic acid and a first variant nucleic acid, wherein the first blocker variable subsequence is complementary to a first variant subsequence of the first variant nucleic acid, and wherein the first blocker oligonucleotide comprises a non-complementary sequence region at its 3′ end, which prevents enzymatic extension and which is not complementary to a sequence continuous with the first target-neutral subsequence;
• (b) a first primer oligonucleotide sufficient to induce enzymatic extension, wherein the first primer oligonucleotide comprises a second sequence that is complementary to a second portion of a homologous subsequence of both the first target nucleic acid and the first variant nucleic acid, wherein the second sequence overlaps the target-neutral subsequence by at least 5 nucleotides such that the second sequence comprises a first overlapping subsequence and a first non-overlapping subsequence, and wherein the second sequence does not include the first blocker variable subsequence;
• (c) a second blocker oligonucleotide comprising a fourth sequence comprising a second target-neutral subsequence and a second blocker variable subsequence, wherein the second target-neutral subsequence comprises a first portion and a second portion, wherein the second blocker variable subsequence is flanked on its 3′ end by the first portion of the second target-neutral subsequence and on its 5′ end by the second portion of the second target-neutral subsequence, and is continuous with the first and second portions of the second target-neutral subsequence, wherein the second target-neutral subsequence is complementary to a first portion of a homologous subsequence of both a second target nucleic acid and a second variant nucleic acid, wherein the second blocker variable subsequence is complementary to a second variant subsequence of the second variant nucleic acid, and wherein the second blocker oligonucleotide comprises a non-complementary sequence region at its 3′ end, which prevents enzymatic extension and which is not complementary to a sequence continuous with the second target-neutral subsequence;
• and
• (d) a second primer oligonucleotide sufficient to induce enzymatic extension, wherein the second primer oligonucleotide comprises a third sequence that is complementary to a second portion of a homologous subsequence of both the second target nucleic acid and the second variant nucleic acid, wherein the third sequence overlaps the second target-neutral subsequence by at least 5 nucleotides such that the third sequence comprises a second overlapping subsequence and a second non-overlapping subsequence, and wherein the third sequence does not include the second blocker variable subsequence.