Patent ID: 6828127
Filing Date: 2004-12-07
Classification: C12N,C12Q

Abstract:
A method of amplifying an RNA target sequence in an RNA sample comprising said target sequence, said method comprising:(A) bringing said sample into contact: with a reagent capable of hybridizing with RNA comprising said target sequence, in the absence of deoxyribonucleoside triphosphates, and with an enzymatic system comprising an RNA polymerase, under conditions allowing the hybridization of said reagent with said RNA comprising said target sequence and under conditions allowing the functioning of said RNA polymerase; wherein said reagent contains: (i) a first nucleotide strand comprising: a) a first nucleotide segment capable of playing the role of sense strand of a promoter for said RNA polymerase and b) downstream of said first segment, a second nucleotide segment comprising a sequence capable of hybridizing with a region of said RNA, and (ii) in the hybridized state on the first strand, a second nucleotide strand comprising a third nucleotide segment capable of hybridizing with said first segment so as to form with it a functional double-stranded promoter; and wherein said RNA polymerase (1) is a T7-like phage RNA polymerase and (2) is capable of transcribing an RNA template, in the presence of said reagent hybridized with said template, in the absence of associated protein factor and in the absence of a ligase activity; and (B) amplifying said target sequence by transcription under control of the double-stranded promotor.