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Fork PCR: a universal and efficient genome-walking tool

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 implem...

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Autores principales: Pan, Hao, Guo, Xinyue, Pan, Zhenkang, Wang, Rongrong, Tian, Bingkun, Li, Haixing
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10556450/
https://www.ncbi.nlm.nih.gov/pubmed/37808312
http://dx.doi.org/10.3389/fmicb.2023.1265580
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author Pan, Hao
Guo, Xinyue
Pan, Zhenkang
Wang, Rongrong
Tian, Bingkun
Li, Haixing
author_facet Pan, Hao
Guo, Xinyue
Pan, Zhenkang
Wang, Rongrong
Tian, Bingkun
Li, Haixing
author_sort Pan, Hao
collection PubMed
description 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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spelling pubmed-105564502023-10-07 Fork PCR: a universal and efficient genome-walking tool Pan, Hao Guo, Xinyue Pan, Zhenkang Wang, Rongrong Tian, Bingkun Li, Haixing Front Microbiol Microbiology 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. Frontiers Media S.A. 2023-09-22 /pmc/articles/PMC10556450/ /pubmed/37808312 http://dx.doi.org/10.3389/fmicb.2023.1265580 Text en Copyright © 2023 Pan, Guo, Pan, Wang, Tian and Li. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Pan, Hao
Guo, Xinyue
Pan, Zhenkang
Wang, Rongrong
Tian, Bingkun
Li, Haixing
Fork PCR: a universal and efficient genome-walking tool
title Fork PCR: a universal and efficient genome-walking tool
title_full Fork PCR: a universal and efficient genome-walking tool
title_fullStr Fork PCR: a universal and efficient genome-walking tool
title_full_unstemmed Fork PCR: a universal and efficient genome-walking tool
title_short Fork PCR: a universal and efficient genome-walking tool
title_sort fork pcr: a universal and efficient genome-walking tool
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10556450/
https://www.ncbi.nlm.nih.gov/pubmed/37808312
http://dx.doi.org/10.3389/fmicb.2023.1265580
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