SINGLE-PRIMER NUCLEIC ACID AMPLIFICATION METHODS

摘要

A method of synthesizing multiple copies of a target sequence, said method comprising the steps of (A) treating a target nucleic acid comprising an RNA, target sequence with: (1) 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.; and (2) a binding molecule which binds to said target nucleic acid adjacent to or near the 5'-end of said target sequence; (B) extending said priming oligonucleotide in a primer extension reaction with a DNA polymerase to give a DNA primer extension product complementary to said target sequence, said DNA primer extension product having a 3'-end which is determined by said binding molecule and which is complementary to the 5'-end of said target sequence; (C) separating said DNA primer extension product from said target sequence using an enzyme which selectively degrades said target sequence; (D) treating said DNA primer extension product with a promoter oligonucleotide comprising first and second regions, said first region hybridizing to a 3'-region of said DNA primer extension product to form a promoter oligonucleotide:DNA primer extension product hybrid, and said second region being 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; (E) extending the 3'-end of said DNA primer extension product in said promoter oligonucleotide:DNA primer extension product hybrid to add a sequence complementary to said second region of said promoter oligonucleotide; and (F) transcribing from said promoter oligonucleotide:DNA primer extension product hybrid multiple RNA products complementary to said DNA primer extension product using an RNA polymerase which recognizes said promoter and initiates transcription therefrom, wherein the base sequences of said RNA products are substantially identical to the base sequence of said target sequence.