Patent ID: 8673567
Filing Date: 2014-03-18
Classification: C12Q

Abstract:
1. A method of detecting the presence of a target sequence in a polynucleotide analyte contained in a sample, comprising: (a) contacting a sample containing an unamplified polynucleotide analyte with a single-stranded DNA target probe having a sequence capable of hybridizing with a target sequence in the polynucleotide analyte, under conditions effective to form a double-stranded complex between the polynucleotide analyte and the single-stranded DNA target probe, wherein the single-stranded DNA target probe has a hairpin structure; (b) incubating the double-stranded complex in the presence of a polymerase and no more than three kinds of nucleotide triphosphates, wherein a selected one or more target-directed nucleotide bases is added to the single-stranded DNA target probe's 3′ end to produce a modified single-stranded DNA target probe having a hairpin structure and a 3′ sticky end; (c) hybridizing the modified single-stranded DNA target probe with a single-stranded DNA detection probe, wherein the single-stranded DNA detection probe (i) does not hybridize to the target sequence in the polynucleotide analyte, (ii) does not ligate with the unmodified single-stranded DNA target probe, and (iii) has a hairpin structure with a 3′ sticky end complementary to the 3′ sticky end of the modified single-stranded DNA target probe; (d) ligating the modified single-stranded DNA target probe and the single-stranded DNA detection probe to form a circular two-probe ligation product, wherein the polynucleotide analyte is not used as a template to form the circular two-probe ligation product; (e) after formation of the circular two-probe ligation product, degrading linear nucleic acid molecules; (f) amplifying the circular two-probe ligation product to produce a detectable amplicon; and (g) detecting the presence of the circular two-probe ligation product by detecting the amplicon.