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

J Microbiol Methods. 2005 Mar;60(3):291-8. doi: 10.1016/j.mimet.2004.10.009.

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.

Publication types

  • Comparative Study

MeSH terms

  • Base Sequence
  • Cloning, Molecular
  • DNA Primers / genetics
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Electrophoresis, Polyacrylamide Gel
  • Fluoresceins / chemistry
  • Genetic Techniques*
  • Helicobacter pylori / genetics*
  • Humans
  • Molecular Sequence Data
  • Promoter Regions, Genetic / genetics*
  • RNA, Bacterial / chemistry
  • RNA, Bacterial / genetics
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Analysis, DNA
  • Transcription, Genetic / genetics

Substances

  • DNA Primers
  • DNA, Bacterial
  • Fluoresceins
  • RNA, Bacterial
  • RNA, Messenger
  • 6-carboxyfluorescein