Patent ID: 6030774
Filing Date: 2000-02-29
Classification: C12Q

Abstract:
A method for detecting the presence of a polynucleotide analyte, said analyte comprising a template sequence having three sequences T1, T2 and T3 wherein T1 is non-contiguous with and 3' of said T2 and T3 and the 5' end of said T3 is 5' of the 5' end of said T2, in a medium suspected of containing said analyte said method comprising:(a) combining said medium with (1) a primer polynucleotide whose 3' end is hybridizable with said T1, (2) a blocker polynucleotide with sequence B1, said B1 being hybridizable with said T3, (3) a polynucleotide Q having sequences S1 and S2 wherein Q is attached at its 3'-end to the 5' end of said blocker polynucleotide or is present as a separate reagent and wherein S1 is 3' of S2 and is substantially identical to said T2 and wherein said S2 is substantially identical to at least the 3' end of said primer polynucleotide, (4) DNA polymerase and (5) deoxynucleoside triphosphates under conditions wherein: (A) said blocker becomes hybridized to said template, (B) said primer becomes hybridized with and extended along, by means of a polymerase, said template and along at least a portion of said T2 and thereafter along said polynucleotide Q to form a duplex, wherein said polymerase has little or no 5'-3' exonuclease activity under said conditions for extending and wherein the 3'-end of said polynucleotide Q, when present as a separate reagent, is not extended by said polymerase, (C) said extended primer is dissociated from the said duplex, and (D) said primer hybridizes with and is extended along said extended primer to form a duplex comprising extended primer and steps (C) and (D) are repeated, and(b) examining for the presence of said extended primer.