Simultaneous detection of DNA and RNA by differential polymerase chain reaction (DIFF-PCR).

Abstract

A new technique, the differential polymerase chain reaction (DIFF-PCR), allows the simultaneous amplification of DNA and homologous RNA in a single assay by the combination of DNA-PCR and RNA-PCR on the same target. DNA-PCR amplifies a selected segment of dsDNA, whereas RNA-PCR amplifies a complementary DNA (cDNA), produced by reverse transcription of RNA. In a mixture of target DNA and RNA, DNA is amplified using a combination of sense and antisense primers under high-stringency conditions giving a D-amplicon. RNA is first reverse-transcribed with a primer carrying a nontarget 5' end into a tagged cDNA at low stringency. Tagged cDNA is subsequently amplified, providing an R-amplicon smaller in size than the D-amplicon. By quantifying the relative amounts of amplified RNA and homologous DNA, a sensitive measure for the transcription rate of a defined DNA segment is obtained. Thus, DIFF-PCR may serve as a useful tool for monitoring gene expression as well as for studying gene regulation and gene function.