Patent ID: 6365346
Filing Date: 2002-04-02
Classification: C12Q

Abstract:
A method for detecting the amount of a target polynucleotide in a sample, said method comprising:(a) providing in single reaction medium (i) a sample suspected of containing said target polynucleotide, said target polynucleotide being in single stranded form, (ii) predetermined amounts of one or more reference polynucleotides, each of said reference polynucleotides comprising a first sequence that is common with a first sequence of said target polynucleotide and a second sequence that is different from a second sequence of said target polynucleotide, (iii) a predetermined amount of a first oligonucleotide probe that has a sequence that hybridizes with said sequence that is common, (iv) a second oligonucleotide probe that has a sequence that hybridizes only with said second sequence of said target polynucleotide, and (v) one or more third oligonucleotide probes, each of said third oligonucleotide probes having a sequence that hybridizes only with a respective second sequence of one of said reference polynucleotide, (b) subjecting said single reaction medium to isothermal conditions for amplifying with equal efficiency said target polynucleotide and said one or more reference polynucleotides, wherein said first probe is not extended, and wherein said conditions permit formation of a substantially non-dissociative first termolecular complex of said target polynucleotide, said first oligonucleotide probe and said second oligonucleotide probe and a substantially non-dissociative second termolecular complex of each of said reference polynucleotide with said first oligonucleotide probe and a respective third oligonucleotide probe and wherein said predetermined amount of said first oligonucleotide probe is less than the expected amount of said amplified target polynucleotide, (c) determining the ratio of the amount of said first termolecular complex to the amount of each of said second termolecular complexes, and (d) relating each of said ratios to the predetermined amount of each of said reference polynucleotides to determine the amount of said target polynucleotide in said sample.