Center Degenerated Walking-Primer PCR: A Novel and Universal Genome-Walking Method.

Enhancing the specificity and applicability of PCR-based genome-walking methods is highly desirable. A new and universal genome-walking tool, called center degenerated walking-primer PCR (CDWP-PCR), is presented in this study. CDWP-PCR involves adopting a center degenerated walking primer (cdWP) in the secondary/tertiary round of amplification. This cdWP is generated by degenerating the seven central nucleotides of the normal walking primer (nWP) used in primary PCR to NNNNNNN (where N includes the bases A, T, C, and G). Clearly, a partially complementary structure is formed between the two primers. Accordingly, the primary CDWP-PCR non-target products defined by the nWP are diluted in secondary/tertiary CDWP-PCR, as these non-targets have difficulty in annealing with the cdWP; conversely, the primary target product can still be efficiently amplified. The working performance of the proposed CDWP-PCR is verified through cloning of the unknown flanks of three known genes. All the clear DNA bands in the tertiary CDWP-PCRs are confirmed to be correct, and the largest DNA band is 8.0 kb. Overall, CDWP-PCR can be considered as a reliable supplement to existing genome-walking methods.