Protocol to clone unknown flanking genomic region using SWPOP-PCR for genome walking

Genome walking is a molecular technique for mining unknown flanking DNAs, significantly advancing fields related to biology. We here detail a genome-walking protocol for stepwise partially overlapping primer-based PCR (SWPOP-PCR). We specify the rules for designing walking primers and gene-specific primers. We also delineate the three-stage workflow of SWPOP-PCR for cloning target DNA, as well as the subsequent purification, sequencing, and analysis of target amplicon. For complete details on the use and execution of this protocol, please refer to Chang et al.¹.