TY - JOUR
T1 - Identifying cloned Helicobacter pylori promoters by primer extension using a FAM-labelled primer and GeneScan® analysis
AU - Lloyd, A.L.
AU - Marshall, Barry
AU - Mee, Brian
PY - 2005
Y1 - 2005
N2 - 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.
AB - 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.
U2 - 10.1016/j.mimet.2004.10.009
DO - 10.1016/j.mimet.2004.10.009
M3 - Article
C2 - 15649531
SN - 0167-7012
VL - 60
SP - 291
EP - 298
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
IS - 3
ER -