Method For Amplification Of Rna Sequences

Method for amplification of a target RNA sequence comprising the following steps:

• (a) annealing a first primer to the target RNA sequence, said first primer comprising a hybridizing sequence, which is complementary to and hybridizes to at least a first segment of the target RNA sequence, operatively associated with a promoter sequence;
• (b) extending said first primer in a reaction catalyzed by a DNA polymerase, forming a first RNA/cDNA hybrid nucleic acid molecule;
• (c) selectively removing the target RNA sequence of the first RNA/cDNA hybrid nucleic acid molecule forming a first single stranded cDNA sequence;
• (d) annealing a second primer to the obtained first single stranded cDNA sequence, said second primer comprising a hybridizing sequence which is complementary to and hybridizes to a first segment of the first single stranded cDNA sequence;
• (e) extending said second primer in a reaction catalyzed by a DNA polymerase to form a first double stranded DNA molecule;
• and
• (f) employing the first double stranded DNA molecule of step (e) in the preparation of a plurality of RNA transcripts that are complementary to the target RNA sequence in a reaction catalyzed by a DNA-dependent RNA polymerase with specificity for the promoter sequence comprised in the first primer;
• wherein the first primer comprises a hybridizing sequence of 7 to 14 nucleotides, a transcription enhancing sequence, and an anchor which is capable of binding to a second segment of the target RNA sequence, and/or wherein the second primer comprises a hybridizing sequence of 7 to 14 nucleotides, an amplification enhancing sequence and an anchor which is capable of binding to a second segment of the first single stranded cDNA.