Uracil walking primer PCR: An accurate and efficient genome-walking tool

Genome walking PCR has been extensively used to acquire unknown genomic regions bordering known DNAs. However, non-target amplification challenges the efficacy of existing genome-walking PCRs. Herein, we conceived a new genome-walking method termed Uracil walking Primer PCR (UP-PCR). The UP-PCR features introducing an uracil base at the penultimate position of arbitrary walking primer (AWP) 3' end. A UP-PCR set comprises three nested amplification steps, which are performed by an AWP sequentially coupling a set of three nested site-specific primers, respectively. Prior to secondary UP-PCR, primary UP-PCR product is processed with uracil DNA glycosylase to destroy the carried AWP. As a result, only target primary product is exponentially amplified in the next UP-PCR(s), as it is the only product with binding sites for the both primers. The performance of UP-PCR has been validated by walking three selected genes. The walking experiments showed that each secondary or tertiary UP-PCR generated one to two amplicon ranging in size from 0.2 to 5.0 kb, while with a negligible non-target background; and the amplicons of the secondary UP-PCRs were all correct, indicating that tertiary UP-PCR is generally unnecessary. These findings suggested that UP-PCR has a satisfactory walking ability, specificity, and speed. Collectively, the proposed UP-PCR is a potential candidate method for genome walking.