Patent ID: 7794986

Claim:
A 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 first hybridizing sequence, comprising 7-14 nucleotides, which is complementary to at least a first segment of the target RNA sequence; a transcription enhancing sequence that comprises a promoter sequence that is operatively associated with the first hybridizing sequence; and a first oligonucleotide anchor that binds to a second segment of the target RNA sequence, wherein the transcription enhancing sequence forms a loop between the first oligonucleotide anchor and the first hybridizing sequence upon annealing of said first primer to the target RNA sequence; (b) extending said first primer in a reaction catalyzed by a DNA polymerase, forming a first RNA/cDNA hybrid nucleic acid molecule; (c) removing the target RNA sequence of the first RNA/cDNA hybrid nucleic acid molecule to obtain a first single stranded cDNA sequence; (d) annealing a second primer to the first single stranded cDNA sequence, said second primer comprising an amplification enhancing sequence having no promoter sequence and a second hybridizing sequence which is complementary 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) amplifying the first double stranded DNA molecule of step (e) using a DNA-dependent RNA polymerase with specificity for said promoter sequence of said first primer to produce a plurality of RNA transcripts that are complementary to the target RNA sequence.