Patent ID: 6379932
Filing Date: 2002-04-30
Classification: C12N,C12Q,Y10T

Abstract:
A method for amplifying a RNA sequence, comprising the steps of:(a) hybridizing to a target RNA a first primer comprising a 3â€² target RNA hybridizing sequence and a first 5â€² defined amplifiable sequence; (b) extending the first primer with a reverse transcriptase to form a first cDNA strand; (c) hybridizing to the first cDNA strand a second primer comprising a 3â€² random cDNA hybridizing sequence and a second 5â€² defined amplifiable sequence; (d) extending the second primer with a DNA polymerase to form a second cDNA stand; and (e) amplifying the second cDNA strand by a polymerase chain reaction with a third primer comprising the first 5â€² defined amplifiable sequence, and a fourth primer comprising the second 5â€² defined amplifiable sequence; wherein step (b) yields a heteroduplex of the target RNA and the first cDNA and further comprises the step of digesting the target RNA of the heteroduplex with a RNAse sufficient to permit hybridization of the first cDNA strand with the second primer without a melting step.