Patent ID: 8137911
Filing Date: 2012-03-20
Classification: C12N

Abstract:
1. A method for making a transcription product having a sequence that is identical to at least a portion of a target sequence in a target nucleic acid consisting of RNA, the method comprising the steps of: (a) providing a sense promoter primer, the sense promoter primer exhibiting, at its 3′-end, a sequence complementary to the 3′-end of the target sequence, the sense promoter primer further exhibiting, at its 5′-end, a sense transcription promoter sequence; (b) annealing the sense promoter primer to the target nucleic acid and primer-extending the sense promoter primer with an RNA-dependent DNA polymerase using target nucleic acid as a template to generate linear sense-promoter-containing first-strand cDNA; (c) removing the target nucleic acid; (d) ligating the first-strand cDNA to itself with a ligase, whereby the 5′-end is covalently joined to the 3′-end to obtain circular first-strand cDNA containing the sense transcription promoter sequence; (e) obtaining a circular transcription substrate; and (f) admixing the transcription substrate with an RNA polymerase and NTPs and incubating under conditions wherein a transcription product having a sequence that is identical to at least a portion of a target sequence is synthesized.