Identifying cloned Helicobacter pylori promoters by primer extension using a FAM-labelled primer and GeneScan analysis.
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.
Lloyd
Amanda L
AL
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.
Marshall
Barry J
BJ
Mee
Brian J
BJ
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005452:Fluoresceins / Q000737:chemistry; D005821:Genetic Techniques; D016480:Helicobacter pylori / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial; D005452:Fluoresceins; D012329:RNA, Bacterial; D012333:RNA, Messenger; C024098:6-carboxyfluorescein