ArticleMolecular biotechnology2011
A novel and simple PCR walking method for rapid acquisition of long DNA sequence flanking a known site in microbial genome.
Article in Molecular biotechnology, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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13 citing papers in PubMed, 14 citations in OpenAlex.
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- DegS protease regulates the motility, chemotaxis, and colonization ofFrontiers in microbiology · 2023Article
- DegS protease regulates antioxidant capacity and adaptability to oxidative stress environment inFrontiers in cellular and infection microbiology · 2023Article
- A Simple Genome Walking Strategy to Isolate Unknown Genomic Regions Using Long Primer and RAPD Primer.Iranian journal of biotechnology · 2019Article
- Exploration of the effects of aPeerJ · 2019Article
- Stepwise partially overlapping primer-based PCR for genome walking.AMB Express · 2018Article
- Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites.AMB Express · 2017Article
- A simple one-step PCR walking method and its application of bacterial rRNA for sequencing identification.Current microbiology · 2014Article
- A simple one-step PCR walking method and its application of bacterial rRNA for sequencing identification.Current microbiology · 2014Article
- High frequency of a novel filamentous phage, VCY φ, within an environmental Vibrio cholerae population.Applied and environmental microbiology · 2012Article
- Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning.BMC biotechnology · 2011Article
- Article
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Authors and funding
4 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Acquisition of flanking sequence adjacent to a known DNA site is an important task in microbial genome-related research. In this study, we developed a new method containing two rounds of PCR followed by cloning and sequencing. Firstly, specific primer (SP) is added into the reaction system for primary locus-specific linear amplification, and then a complex long primer (CLP) is added into the cooled reaction system for only one cycle. Amplification products from the first round of PCR are directly purified without electrophoresis, diluted, and used as the templates of the second PCR. Secondly, one long specific primer (LSP) and one long base-fixed primer (LFP) are adopted. The amplicons are purified for cloning and sequencing. The achievement of specific amplification for long flanking region mainly depends on ingenious and precise settings of PCR programs, structure design of CLP primer, adding of CLP primer after specific linear amplification, concentration ratio of CLP and SP primer, applying long primers, etc. Through this method, we successfully performed the long PCR walkings (>1.5 Kb) on rpoB gene of Vibrio vulnificus, transposon-like gene of V. alginolyticus, and sto gene of V. cholerae. The method provides a robust and simple strategy for rapid amplification of long unknown DNA fragments from microbes.
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