Protocol to access unknown flanking DNA sequences using Wristwatch-PCR for genome-walking

• Published in: STAR protocols 2023-03, Vol.4 (1), p.102037-102037, Article 102037
• Main Authors: Wang, Lingqin, Jia, Mengya, Li, Zhaoqin, Liu, Xiaohua, Sun, Tianyi, Pei, Jinfeng, Wei, Cheng, Lin, Zhiyu, Li, Haixing
• Format: Article
• Language: English
• Subjects: Biotechnology and bioengineering; Genetics; Molecular Biology; Protocol; Sequencing
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.102037

Here we describe a protocol for wristwatch PCR, an approach based on wristwatch-like structure formed between walking primers to obtain unknown flanks. We specify the criteria for designing wristwatch primers and gene-specific primers. We detail how to set wristwatch primer permutations to obtain personalized walking outcomes and improve walking efficiency. We describe experimental procedures for isolating a DNA of interest using three rounds of nested wristwatch PCR as well as the subsequent steps for DNA purification, cloning, and sequencing. For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).1 [Display omitted] •Design of a wristwatch primer set to selectively enrich target DNA•Parallel use of wristwatch primer permutations to ensure walking efficiency•Conduct three successive nested PCRs implemented for genome-walking Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here we describe a protocol for wristwatch PCR, an approach based on wristwatch-like structure formed between walking primers to obtain unknown flanks. We specify the criteria for designing wristwatch primers and gene-specific primers. We detail how to set wristwatch primer permutations to obtain personalized walking outcomes and improve walking efficiency. We describe experimental procedures for isolating a DNA of interest using three rounds of nested wristwatch PCR as well as the subsequent steps for DNA purification, cloning, and sequencing.