Gw. Buchman et al., SELECTIVE RNA AMPLIFICATION - A NOVEL METHOD USING DUMP-CONTAINING PRIMERS AND URACIL DNA GLYCOSYLASE, PCR methods and applications, 3(1), 1993, pp. 28-31
The application of PCR to a wide variety of biological problems and mo
lecular techniques has gained wide acceptance. RNA-PCR, a technique in
which first-strand cDNA synthesis is followed by PCR amplification, h
as enabled detection and characterization of rare transcripts. One pro
blem confronting the researcher involves specific amplification of tra
nscribed sequences in the presence of small amounts of genomic DNA of
identical sequence. We describe a novel technique, selective RNA ampli
fication, which will specifically amplify RNA sequences in a backgroun
d of homologous DNA. The method involves first-strand cDNA synthesis f
rom a specific dUMP-containing oligonucleotide that contains unique us
er-defined 5' sequence (adapter sequence) not found in the message of
interest. RNA template is degraded using RNase H, which is specific fo
r RNA/DNA hybrids. This is followed by second-strand synthesis using a
gene-specific primer (GSP). The original adapter primer is digested w
ith uracil DNA glycosylase (UDG) to prevent its participation in subse
quent amplification. PCR is then performed using the GSP and a second
primer corresponding to the unique adapter sequence. In this paper, we
apply this method to the amplification of RNA derived from human papi
lloma virus sequences. Using Southern analysis, we demonstrate specifi
c amplification of 10(5) molecules of an in vitro-transcribed RNA. Den
atured DNA of identical sequence and concentration was not amplified u
sing the RNA-specific method. The method could eliminate the need for
stringent purification of RNA and enables amplification of rare messag
es from RNA preparations containing homologous DNA of identical sequen
ce and size.