SELECTIVE RNA AMPLIFICATION - A NOVEL METHOD USING DUMP-CONTAINING PRIMERS AND URACIL DNA GLYCOSYLASE

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.