Use of G-clamp for improved allele-specific PCR

摘要

The present invention includes a method of allele-specific amplification, utilizing an allele-specific oligonucleotide, at least partially complementary to more than one variant of the target sequence, but having at least one selective nucleotide complementary to only one variant of the target sequence and incorporating at least one “G-clamp” nucleotide.

主权项

1. A method of allele-specific amplification of a variant of a target sequence, the target existing in the form of several variant sequences, the method comprising:
(a) hybridizing a first and a second oligonucleotides to at least one variant of the target sequence; wherein the first oligonucleotide is at least partially complementary to one or more variants of the target sequence, and the second oligonucleotide is at least partially complementary to one or more variants of the target sequence, and has at least one selective nucleotide at the 3′ terminal nucleotide that is complementary to only one variant of the target sequence; wherein said second oligonucleotide incorporates at least one G-clamp nucleotide at a position between 1 and 5 nucleotide upstream of the 3′ terminal nucleotide; (b) extending the second oligonucleotide with a nucleic acid polymerase, wherein said polymerase is capable of extending said second oligonucleotide efficiently when said second oligonucleotide is hybridized to a variant of the target sequence which is complementary to the one selective nucleotide at the 3′ terminus, and substantially less efficiently when said second oligonucleotide is hybridized to a variant of the target sequence which is not complementary to the one selective nucleotide at the 3′ terminus.