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. employs primer set of three random oligomers to implement walking task. In primary PCR, the low-stringency amplification cycle mediates binding places on genome, producing batch single-stranded DNAs. subsequent high-stringency amplification, single-strand is processed into double-strand by site-specific primer, but non-target DNA cannot be any primer. As result, only can exponentially amplified in remaining cycles. Secondary/tertiary nested PCR(s) further magnifies difference between both DNAs selectively enriching DNA. applicability validated several gene loci. could perspective substitution existing schemes.