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

A.L. Lloyd, Barry Marshall, Brian Mee

Research output: Contribution to journalArticle

47 Citations (Scopus)

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((R)) 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. (C) 2004 Elsevier B.V. All rights reserved.
Original languageEnglish
Pages (from-to)291-298
JournalJournal of Microbiological Methods
Volume60
Issue number3
DOIs
Publication statusPublished - 2005

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