State Key Laboratory of Food Science and Resource, Nanchang University, Nanchang, 330047, China; International Institute of Food Innovation Co., Ltd., Nanchang University, Nanchang, 330020, China; Sino-German Joint Research Institute, Nanchang University, Nanchang, 330047, China.
State Key Laboratory of Food Science and Resource, Nanchang University, Nanchang, 330047, China; International Institute of Food Innovation Co., Ltd., Nanchang University, Nanchang, 330020, China; Sino-German Joint Research Institute, Nanchang University, Nanchang, 330047, China.
Anal Biochem. 2025 Jan;696:115697. doi: 10.1016/j.ab.2024.115697. Epub 2024 Oct 22.
PCR-based DNA walking is of efficacy for capturing unknown flanking genomic sequences. Here, an uracil base PCR (UB-PCR) with satisfying specificity has been devised for DNA walking. Primary UB-PCR replaces thymine base with uracil base, resulting in a primary PCR product composed of U-DNAs. A single-primer (primary nested sequence-specific primer) single-cycle amplification, using the four normal bases (adenine, thymine, cytosine, and guanine) as substrate, is then performed on the primary PCR product. Clearly, only those U-DNAs, ended by the primary nested sequence-specific primer at least at one side, will produce the corresponding normal single strands. Next, the single-cycle product undergoes uracil-DNA glycosylase treatment to destroy the U-DNAs, while the normal single strands are unaffected. Afterward, secondary even tertiary PCR is performed to exclusively enrich the target product. The feasibility of UB-PCR has been checked by obtaining unknown sequences bordering the three selected genetic sites.
基于 PCR 的 DNA 游走可有效地捕获未知侧翼基因组序列。在此，设计了一种具有令人满意特异性的尿嘧啶基 PCR（UB-PCR）用于 DNA 游走。初级 UB-PCR 用尿嘧啶碱基替代胸腺嘧啶碱基，从而产生由 U-DNA 组成的初级 PCR 产物。然后，使用四种正常碱基（腺嘌呤、胸腺嘧啶、胞嘧啶和鸟嘌呤）作为底物，对初级 PCR 产物进行单引物（初级嵌套序列特异性引物）单循环扩增。显然，只有那些至少在一侧由初级嵌套序列特异性引物结束的 U-DNAs，才会产生相应的正常单链。接下来，单循环产物经过尿嘧啶-DNA 糖基化酶处理以破坏 U-DNAs，而正常单链不受影响。之后，进行二次甚至三次 PCR 以专门富集目标产物。通过获得三个选定遗传位点周围的未知序列，验证了 UB-PCR 的可行性。
