Chen Hong, Tian Bingkun, Wang Rongrong, Pan Zhenkang, Gao Dandan, Li Haixing
State Key Laboratory of Food Science and Resource, Nanchang University, Nanchnag, China; International Institute of Food Innovation Co., Ltd., Nanchang University, Nanchnag, China; Sino-German Joint Research Institute, Nanchang University, Nanchang, China.
State Key Laboratory of Food Science and Resource, Nanchang University, Nanchnag, China; Sino-German Joint Research Institute, Nanchang University, Nanchang, China.
J Genet Eng Biotechnol. 2025 Jun;23(2):100478. doi: 10.1016/j.jgeb.2025.100478. Epub 2025 Mar 13.
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.
基因组步移PCR已被广泛用于获取与已知DNA相邻的未知基因组区域。然而,非靶向扩增对现有基因组步移PCR的效率提出了挑战。在此,我们构思了一种新的基因组步移方法,称为尿嘧啶步移引物PCR(UP-PCR)。UP-PCR的特点是在任意步移引物(AWP)3'端的倒数第二个位置引入一个尿嘧啶碱基。一个UP-PCR组包括三个嵌套扩增步骤,分别由AWP依次与一组三个嵌套的位点特异性引物偶联进行。在二次UP-PCR之前,用尿嘧啶DNA糖基化酶处理一次UP-PCR产物以破坏携带的AWP。结果,在下一次UP-PCR中,只有目标一次产物呈指数扩增,因为它是唯一具有两个引物结合位点的产物。通过对三个选定基因进行步移验证了UP-PCR的性能。步移实验表明,每个二次或三次UP-PCR产生1至2个大小在0.2至5.0 kb之间的扩增子,而非靶向背景可忽略不计;二次UP-PCR的扩增子均正确,表明通常不需要三次UP-PCR。这些发现表明,UP-PCR具有令人满意的步移能力、特异性和速度。总的来说,所提出的UP-PCR是一种潜在的基因组步移候选方法。