Patent ID: 9238832
Filing Date: 2016-01-19
CPC Classification: C12Q

Claim Text:
1. A method of allele-specific amplification by polymerase chain reaction (PCR) and detection of a desired variant of a target nucleic acid, the target nucleic acid existing in the form of several variant sequences, the method comprising: (a) providing a reaction mixture in liquid solution that comprises a first oligonucleotide at least partially complementary to one or more variants of the target nucleic acid, and a second oligonucleotide at least partially complementary to one or more variants of the target nucleic acid and has at least one selective nucleotide that is complementary to a corresponding nucleotide in the desired variant of the target nucleic acid but not complementary to the corresponding nucleotide in undesired variants of the target nucleic acid, wherein said selective nucleotide is placed internally of the 3′ terminus, and wherein said second oligonucleotide further comprises at least one nucleotide with a base covalently modified at the exocyclic amino group; (b) hybridizing said first and second oligonucleotides to at least one variant of the target nucleic acid; (c) providing conditions for the extension of said first and second oligonucleotides to at least one variant of the target nucleic acid by a nucleic acid polymerase, wherein said polymerase is capable of extending said second oligonucleotide preferentially when said selective nucleotide forms a base pair with the desired variant of the target nucleic acid, and substantially less when said selective nucleotide does not form a base pair with the desired variant of the target nucleic acid; and (d) detecting the product of primer extension in step (c); wherein said method of amplification and detection are performed in a homogeneous assay.