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

Citation
D. Tillett et al., Optimized rapid amplification of cDNA ends (RACE) for mapping bacterial mRNA transcripts, BIOTECHNIQU, 28(3), 2000, pp. 448
Citations number
10
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOTECHNIQUES
ISSN journal
07366205 → ACNP
Volume
28
Issue
3
Year of publication
2000
Database
ISI
SICI code
0736-6205(200003)28:3<448:ORAOCE>2.0.ZU;2-1
Abstract
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.