Optimized rapid amplification of cDNA ends (RACE) for mapping bacterial mRNA transcripts

A simple, efficient and sensitive RACE-based procedure was developed for th e determination of unknown 5' regions from bacterial cDNA. A number of crit ical modifications were made to the standard RACE method including the opti mization of the RNA extraction, reverse transcription and PCR conditions. T his procedure was used to accurately determine the site of transcript initi ation and structure of the promoter region of the Helicobacter pylori aspar tate carbamoyltransferase gene (pyrB). The technique avoids many of the dif ficulties associated with established bacterial transcript mapping protocol s and can be performed in two days starting with less than 1 mu g of total RNA. The modifications described here have significant potential for the id entification of transcript start sites of bacterial genes and non-polyadeny lated eukaryotic RNA.