Synthesis of RNA probes by the direct in vitro transcription of PCR-generated DNA templates.
We describe a novel method for the generation of RNA probes based on the direct in vitro transcription of DNA templates amplified by polymerase chain reaction (PCR) using primers with sequence hybrids between the target gene and those of the T7 and T3 RNA polymerases promoters. This method circumvents the need for cloning and allows rapid generation of strand-specific RNA molecules that can be used for the identification of genes in hybridization experiments. We have successfully applied this method to the identification of DNA sequences by Southern blot analysis and library screening.
Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892.
R
R
Urrutia
M A
MA
McNiven
B
B
Kachar
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D015347:RNA Probes / Q000096:biosynthesis*; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins
D015347:RNA Probes; D014764:Viral Proteins; D004247:DNA; C081591:bacteriophage T3 RNA polymerase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases