ArticleFrontiers in microbiology2023
Fork PCR: a universal and efficient genome-walking tool.
Article in Frontiers in microbiology, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed.
- Article
- Bridging PCR-Based Genome-Walking Protocol.Bio-protocol · 2025Article
- NBiochemical genetics · 2025Article
- Uracil walking primer PCR: An accurate and efficient genome-walking tool.Journal, genetic engineering & biotechnology · 2025Article
- Transgene Mapping in Animals: What to Choose?International journal of molecular sciences · 2025Review
- Protocol to Mine Unknown Flanking DNA Using PER-PCR for Genome Walking.Bio-protocol · 2025Article
- Protocol to Identify Unknown Flanking DNA Using Partially Overlapping Primer-based PCR for Genome Walking.Bio-protocol · 2025Article
- Protocol to Retrieve Unknown Flanking DNA Using Fork PCR for Genome Walking.Bio-protocol · 2025Article
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Authors and funding
6 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The reported genome-walking methods still suffer from some deficiencies, such as cumbersome experimental steps, short target amplicon, or deep background. Here, a simple and practical fork PCR was proposed for genome-walking. The fork PCR employs a fork primer set of three random oligomers to implement walking task. In primary fork PCR, the low-stringency amplification cycle mediates the random binding of primary fork primer to some places on genome, producing a batch of single-stranded DNAs. In the subsequent high-stringency amplification, the target single-strand is processed into double-strand by the site-specific primer, but a non-target single-stranded DNA cannot be processed by any primer. As a result, only the target DNA can be exponentially amplified in the remaining high-stringency cycles. Secondary/tertiary nested fork PCR(s) further magnifies the amplification difference between the both DNAs by selectively enriching target DNA. The applicability of fork PCR was validated by walking several gene loci. The fork PCR could be a perspective substitution for the existing genome-walking schemes.
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