Patent ID: 6271002
Filing Date: 2001-08-07
Classification: C12N

Abstract:
A method for amplifying at least one mRNA in a sample containing a plurality of different mRNAs comprising:(a) synthesizing first strand cDNA by contacting under conditions conducive to reverse transcriptase activity at least one mRNA in said sample with (i) reverse transcriptase, and (ii) a first primer that is sufficiently complementary to a sequence in the mRNA so as to prime synthesis in a direction toward the 5' end of the mRNA;(b) synthesizing double-stranded cDNA by contacting under conditions conducive to DNA polymerase activity the first strand cDNA with (i) a first DNA polymerase, and (ii) a second primer that is sufficiently complementary to a sequence 5' to said first primer sequence in said first strand cDNA so as to prime synthesis in a direction toward said first primer sequence; wherein neither said first primer nor said second primer comprises an RNA polymerase promoter sequence in sense or antisense orientation;(c) amplifying the double-stranded cDNA by subjecting the double-stranded cDNA to a single round of polymerase chain reaction (hereinafter ""PCR"") of 20 cycles or less, wherein DNA is synthesized by use of a second DNA polymerase and a primer pair comprising a forward primer and a reverse primer, said forward primer and said reverse primer each being sufficiently complementary to a different strand of said double-stranded cDNA so as to prime synthesis in a template-dependent manner, wherein said forward primer or reverse primer comprises an RNA polymerase promoter sequence in sense or antisense orientation; and(d) transcribing resultant amplified DNA into cRNA by contacting the amplified DNA with an RNA polymerase specific for said RNA polymerase promoter sequence introduced in step (c) under conditions conducive to RNA polymerase activity, such that cRNA is produced.