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J Microbiol Methods. 2005 Mar;60(3):291-8.

Identifying cloned Helicobacter pylori promoters by primer extension using a FAM-labelled primer and GeneScan analysis.

Author information

1
Discipline of Microbiology (M502), School of Biomedical and Chemical Sciences, University of Western Australia, Rm 1.11, L Block, QEII Medical Centre, Verdun Street, Nedlands, WA 6009, Australia.

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.

PMID:
15649531
DOI:
10.1016/j.mimet.2004.10.009
[Indexed for MEDLINE]

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