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 molecular techniques has gained wide acceptance. RNA-PCR, a technique in which first-strand cDNA synthesis is followed by PCR amplification, has enabled detection and characterization of rare transcripts. One problem confronting the researcher involves specific amplification of transcribed sequences in the presence of small amounts of genomic DNA of identical sequence. We describe a novel technique, selective RNA amplification, which will specifically amplify RNA sequences in a background of homologous DNA. The method involves first-strand cDNA synthesis from a specific dUMP-containing oligonucleotide that contains unique user-defined 5' sequence (adapter sequence) not found in the message of interest. RNA template is degraded using RNase H, which is specific for RNA/DNA hybrids. This is followed by second-strand synthesis using a gene-specific primer (GSP). The original adapter primer is digested with uracil DNA glycosylase (UDG) to prevent its participation in subsequent 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 papilloma virus sequences. Using Southern analysis, we demonstrate specific amplification of 10(5) molecules of an in vitro-transcribed RNA. Denatured DNA of identical sequence and concentration was not amplified using the RNA-specific method. The method could eliminate the need for stringent purification of RNA and enables amplification of rare messages from RNA preparations containing homologous DNA of identical sequence and size.
Buchman
G W
GW
Life Technologies, Inc., Gibco/BRL, Gaithersburg, Maryland 20884-9980.
Schuster
D M
DM
Rashtchian
A
A
D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers; D004279:DNA, Viral / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis* / Q000096:biosynthesis / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; D019914:Taq Polymerase
D017931:DNA Primers; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D012367:RNA, Viral; D012313:RNA; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H