Optimized rapid amplification of cDNA ends (RACE) for mapping bacterial mRNA transcripts.
A simple, efficient and sensitive RACE-based procedure was developed for the determination of unknown 5' regions from bacterial cDNA. A number of critical modifications were made to the standard RACE method, including the optimization of the RNA extraction, reverse transcription and PCR conditions. This procedure was used to accurately determine the site of transcript initiation and structure of the promoter region of the Helicobacter pylori aspartate carbamoyltransferase gene (pyrB). The technique avoids many of the difficulties associated with established bacterial transcript mapping protocols and can be performed in two days starting with less than 1 microgram of total RNA. The modifications described here have significant potential for the identification of transcript start sites of bacterial genes and non-polyadenylated eukaryotic RNA.
Tillett
D
D
University of New South Wales, Sydney, Australia.
Burns
B P
BP
Neilan
B A
BA
D018076:DNA, Complementary / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012329:RNA, Bacterial / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*; D014158:Transcription, Genetic
D018076:DNA, Complementary; D012329:RNA, Bacterial; D012333:RNA, Messenger