Patent ID: 8642264
Filing Date: 2014-02-04
Classification: C12Q

Abstract:
1. A method of determining which of a target nucleic acid and a reference nucleic acid is present in a greater amount in a sample comprising: a) amplifying a target nucleic acid and a reference nucleic acid by polymerase chain reaction using a forward primer and a reverse primer for the target nucleic acid and a forward primer and a reverse primer for the reference nucleic acid, wherein the reverse primer for the reference nucleic acid comprises a tail sequence at its 5′-end which is identical to a portion of the target nucleic acid sequence; b) adding a first disruption sequence which is complementary to the reverse primer for the target nucleic acid, and adding a second disruption sequence which is complementary to a portion, but not the tail sequence, of the reverse primer for the reference nucleic acid; c) performing one or more PCR cycles, such that products of the PCR include single-stranded copies of the target nucleic acid and single-stranded copies of the reference nucleic acid that incorporate the complement of the tail sequence; d) allowing the complement of the tail sequence to anneal with its complementary sequence on the target nucleic acid to yield a partially double-stranded nucleic acid molecule, a portion of which is extended to a double-stranded nucleic acid product, while any excess target nucleic acid or reference nucleic acid remains single-stranded; e) crosslinking double-stranded nucleic acid in the mixture resulting from step (d); and f) detecting the presence of single-stranded target nucleic acid and reference nucleic acid, wherein the presence of a substantially greater amount of single-stranded target nucleic acid indicates that a greater amount of target nucleic acid was present in the sample, and the presence of a substantially greater amount of single-stranded reference nucleic acid indicates that a greater amount of reference nucleic acid was present in the sample.