Patent ID: 7713697
Filing Date: 2010-05-11
Classification: C12P,C12Q

Abstract:
1. A method of synthesizing multiple copies of a target sequence, said method comprising: treating a target nucleic acid comprising a DNA target sequence with a priming oligonucleotide which hybridizes to the 3′-end of said target sequence such that a primer extension reaction can be initiated therefrom, wherein said priming oligonucleotide does not comprise RNA; extending said priming oligonucleotide in a primer extension reaction with a DNA polymerase to give a first DNA primer extension product, at least a portion of said first primer extension product being complementary to said target sequence; treating said target nucleic acid with a displacer oligonucleotide which hybridizes to said target nucleic acid upstream from said priming oligonucleotide such that a primer extension reaction can be initiated therefrom; extending said displacer oligonucleotide in a primer extension reaction with said DNA polymerase to give a second DNA primer extension product that displaces said first primer extension product from said target nucleic acid; treating said first primer extension product with a promoter oligonucleotide comprising first and second regions, said first region comprising a base sequence which corresponds to a region at the 5′-end of said target sequence and which hybridizes to said first primer extension product to form a promoter oligonucleotide:first primer extension product hybrid, and said second region comprising a promoter for an RNA polymerase and situated 5′ to said first region, wherein said promoter oligonucleotide is modified to prevent the initiation of DNA synthesis therefrom; transcribing from said promoter oligonucleotide:first primer extension product hybrid multiple first RNA products complementary to at least a portion of said first primer extension product using an RNA polymerase which recognizes said promoter and initiates transcription therefrom, wherein the base sequences of said first RNA products are substantially identical to the base sequence of said target sequence, provided that if said first primer extension product has a defined 3′-end, then said method further comprises treating said target nucleic acid with a binding molecule which binds to said target nucleic acid adjacent to or near the 5′-end of said target sequence.