| Literature DB >> 15649531 |
Amanda L Lloyd1, Barry J Marshall, Brian J Mee.
Abstract
The transcriptional start sites of 27 promoters in Helicobacter pylori strain 4187E have been successfully identified using a non-radioactive primer extension protocol. The technique involves reverse transcribing mRNA with a sequence-specific FAM-labelled primer. The length of the FAM-labelled cDNA primer extension product can be analysed on a standard DNA sequencer using GeneScan software. This information can be used in conjunction with DNA sequencing data to identify the transcriptional start site of a promoter. Total bacterial RNA produced more specific primer extension products with stronger FAM signals than a population enriched for mRNA. Using this technology, it is not necessary to complete the DNA sequencing reactions in parallel with the primer extension experiments. The FAM-labelled primer extension products do not require a PCR amplification step prior to analysis on a sequencing gel, and no phenol/chloroform purifications are required at any stage of the procedure. Fluorescent-based primer extension methods have obvious advantages over the conventional radioactive protocols, and this report extends the currently used methodologies in this field.Entities:
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Year: 2005 PMID： 15649531 DOI： 10.1016/j.mimet.2004.10.009
Source DB: PubMed Journal: J Microbiol Methods ISSN： 0167-7012 Impact factor: 2.363