Patent ID: 6270966
Filing Date: 2001-08-07
Classification: C12N,C12Q

Abstract:
A method for characterizing mRNA molecules in a population of mRNAs including the steps of providing purified and isolated mRNA molecules, reverse transcribing a double stranded cDNA from said isolated mRNA molecules, digesting said double stranded cDNA with a restriction endonuclease to produce cDNA fragments in which either one end or both ends of said cDNA fragments have a sequence that specifically hybridizes to adaptor DNA sequences, wherein the improvement comprises:after said digesting step, hybridizing adaptor DNA sequences to said cDNA fragments in which both ends of said cDNA fragments have a sequence that specifically hybridizes to said adaptor DNA sequences;ligating said adaptor DNA sequences to said cDNA fragments having hybridized adaptor DNA sequences;amplifying said cDNA fragments having ligated adaptor DNA sequences by a polymerase chain reaction (PCR) with primer DNA sequences that hybridize to the ends of said cDNA fragments having ligated adaptor DNA sequences, wherein said primer DNA sequences have at least one nucleotide at the 3' end that specifically hybridizes to a subset of cDNA molecules produced during said reverse transcribing step, thereby producing amplified cDNA fragments; anddetecting the presence of the amplified cDNA fragments, wherein each amplified cDNA fragment corresponds to a mRNA molecule within said population of mRNAs, thereby characterizing mRNA molecules in a population of mRNAs.