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Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy

Genome walking is a method used to retrieve unknown flanking DNA. Here, we reported wristwatch (WW) PCR, an efficient genome walking technique mediated by WW primers (WWPs). WWPs feature 5′- and 3′-overlap and a heterologous interval. Therefore, a wristwatch-like structure can be formed between WWPs...

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Autores principales: Wang, Lingqin, Jia, Mengya, Li, Zhaoqin, Liu, Xiaohua, Sun, Tianyi, Pei, Jinfeng, Wei, Cheng, Lin, Zhiyu, Li, Haixing
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9039356/
https://www.ncbi.nlm.nih.gov/pubmed/35497369
http://dx.doi.org/10.3389/fbioe.2022.792848
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author Wang, Lingqin
Jia, Mengya
Li, Zhaoqin
Liu, Xiaohua
Sun, Tianyi
Pei, Jinfeng
Wei, Cheng
Lin, Zhiyu
Li, Haixing
author_facet Wang, Lingqin
Jia, Mengya
Li, Zhaoqin
Liu, Xiaohua
Sun, Tianyi
Pei, Jinfeng
Wei, Cheng
Lin, Zhiyu
Li, Haixing
author_sort Wang, Lingqin
collection PubMed
description Genome walking is a method used to retrieve unknown flanking DNA. Here, we reported wristwatch (WW) PCR, an efficient genome walking technique mediated by WW primers (WWPs). WWPs feature 5′- and 3′-overlap and a heterologous interval. Therefore, a wristwatch-like structure can be formed between WWPs under relatively low temperatures. Each WW-PCR set is composed of three nested (primary, secondary, and tertiary) PCRs individually performed by three WWPs. The WWP is arbitrarily annealed somewhere on the genome in the one low-stringency cycle of the primary PCR, or directionally to the previous WWP site in one reduced-stringency cycle of the secondary/tertiary PCR, producing a pool of single-stranded DNAs (ssDNAs). A target ssDNA incorporates a gene-specific primer (GSP) complementary at the 3′-end and the WWP at the 5′-end and thus can be exponentially amplified in the next high-stringency cycles. Nevertheless, a non-target ssDNA cannot be amplified as it lacks a perfect binding site for any primers. The practicability of the WW-PCR was validated by successfully accessing unknown regions flanking Lactobacillus brevis CD0817 glutamate decarboxylase gene and the hygromycin gene of rice. The WW-PCR is an attractive alternative to the existing genome walking techniques.
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spelling pubmed-90393562022-04-27 Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy Wang, Lingqin Jia, Mengya Li, Zhaoqin Liu, Xiaohua Sun, Tianyi Pei, Jinfeng Wei, Cheng Lin, Zhiyu Li, Haixing Front Bioeng Biotechnol Bioengineering and Biotechnology Genome walking is a method used to retrieve unknown flanking DNA. Here, we reported wristwatch (WW) PCR, an efficient genome walking technique mediated by WW primers (WWPs). WWPs feature 5′- and 3′-overlap and a heterologous interval. Therefore, a wristwatch-like structure can be formed between WWPs under relatively low temperatures. Each WW-PCR set is composed of three nested (primary, secondary, and tertiary) PCRs individually performed by three WWPs. The WWP is arbitrarily annealed somewhere on the genome in the one low-stringency cycle of the primary PCR, or directionally to the previous WWP site in one reduced-stringency cycle of the secondary/tertiary PCR, producing a pool of single-stranded DNAs (ssDNAs). A target ssDNA incorporates a gene-specific primer (GSP) complementary at the 3′-end and the WWP at the 5′-end and thus can be exponentially amplified in the next high-stringency cycles. Nevertheless, a non-target ssDNA cannot be amplified as it lacks a perfect binding site for any primers. The practicability of the WW-PCR was validated by successfully accessing unknown regions flanking Lactobacillus brevis CD0817 glutamate decarboxylase gene and the hygromycin gene of rice. The WW-PCR is an attractive alternative to the existing genome walking techniques. Frontiers Media S.A. 2022-04-12 /pmc/articles/PMC9039356/ /pubmed/35497369 http://dx.doi.org/10.3389/fbioe.2022.792848 Text en Copyright © 2022 Wang, Jia, Li, Liu, Sun, Pei, Wei, Lin 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 Bioengineering and Biotechnology
Wang, Lingqin
Jia, Mengya
Li, Zhaoqin
Liu, Xiaohua
Sun, Tianyi
Pei, Jinfeng
Wei, Cheng
Lin, Zhiyu
Li, Haixing
Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy
title Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy
title_full Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy
title_fullStr Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy
title_full_unstemmed Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy
title_short Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy
title_sort wristwatch pcr: a versatile and efficient genome walking strategy
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9039356/
https://www.ncbi.nlm.nih.gov/pubmed/35497369
http://dx.doi.org/10.3389/fbioe.2022.792848
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