Patent ID: 6338954
Filing Date: 2002-01-15
Classification: C12N,C12Q

Abstract:
A method for creating, in a non-specific manner, multiple RNA copies, comprising the steps of:contacting a nucleic acid containing starting material comprising a pool of mRNAs, wherein said mRNAs comprise a poly-A tail, with i) an oligonucleotide containing a 5â€² end and a 3â€² end, said oligonucleotide comprising an oligo-dT sequence at said 3â€² end, a promoter sequence recognized by a RNA polymerase at said 5â€² end and a transcription initiation region located between the oligo-dT sequence and the promoter sequence, wherein the oligonucleotide is blocked at said 3â€² end such that extension therefrom is prohibited, ii) an enzyme having reverse transcription activity which forms a double stranded promoter-primer sequence, iii) at least one enzyme having RNase H activity, iv) an enzyme having RNA polymerase activity, and v) sufficient amounts of dNTPs and rNTPs, and maintaining the resulting reaction mixture under appropriate conditions for a sufficient amount of time for the enzymatic processes to occur, such that antisense RNA is formed in the absence of cDNA intermediates.