Patent ID: 7785776
Filing Date: 2010-08-31
Classification: C12Q

Abstract:
1. A method for identifying a heterozygote in a nucleic acid sample, comprising a) amplifying the target nucleic acid in a nucleic acid amplification reaction mixture to generate an amplification product, the amplification reaction mixture comprising aa) a polymerase, ab) deoxynucleoside triphosphates or functional analogues, ac) at least a first primer and a second primer, the first primer being sufficiently complementary to the target nucleic acid to hybridize therewith and initiate template dependent synthesis by the polymerase, the second primer being sufficiently complementary to the complement of the target nucleic acid to hybridize therewith and initiate template dependent synthesis by the polymerase, characterized in that at least one primer is labeled with a fluorescent compound which is not in a FRET donor-acceptor relationship with any other fluorescent entity that may be present in the sample, the labeled primer being selected from the group consisting of b) denaturing the amplification product into single strands and subsequently renaturing the single strands to form a double stranded product, said double stranded product comprising duplexes formed between an amplification extended labeled primer and a full length complementary sequence, c) subjecting the renatured amplification mixture containing the amplified target sequence to a double stranded DNA denaturing gradient and simultaneously monitoring fluorescence emission to generate a curve, and d) identifying the heterozygote by analyzing the shape of the curve generated from the melting of duplexes formed between the amplification extended labeled primer and the full length complementary sequence.