N₇-Ended Walker PCR: An Efficient Genome-Walking Tool

As a tool for acquiring uncharacterized genomic DNA adjacent to characterized DNA, genome-walking is integral to bioscience-related research works. Herein, a new genome-walking tool known as N₇-ended walker PCR (polymerase chain reaction) is presented. The key aspect for this method is the use of a degenerate walker primer in secondary/tertiary PCR. The 7 nt 5' tail of this primer completely degenerates to N₇ relative to its corresponding primary walker primer. The degeneracy reduces the efficiency of annealing this primer to its predecessor site. Clearly, primary nontarget DNA defined by the primary walker primer prefers to form a hairpin structure via the inverted ends rather than hybridizing with the degenerate primer. As a result, N₇-ended walker PCR achieves genome-walking by selectively boosting the DNA of interest. The feasibility of the N₇-ended walker PCR method was proven by acquiring uncharacterized DNAs flanking several characterized DNAs.