Simultaneous detection of DNA and RNA by differential polymerase chain reaction (DIFF-PCR).
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.
Imboden
P
P
Institute of Medical Microbiology, University of Berne, Switzerland.
Burkart
T
T
Schopfer
K
K
D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D015870:Gene Expression; D006678:HIV / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral; D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase