Uracil base PCR implemented for reliable DNA walking

• Published in: Analytical biochemistry 2025-01, Vol.696, p.115697, Article 115697
• Main Authors: Wang, Rongrong, Gu, Yinwei, Chen, Hong, Tian, Bingkun, Li, Haixing
• Format: Article
• Language: English
• Subjects: Base Sequence; DNA - analysis; DNA - genetics; DNA Primers - chemistry; DNA Primers - metabolism; DNA walking; Humans; Nested PCR; Polymerase Chain Reaction - methods; Random annealing; Uracil - chemistry; Uracil - metabolism; Uracil base; Uracil-DNA Glycosidase - genetics; Uracil-DNA Glycosidase - metabolism; Uracil-DNA glycosylase; Walking primer
• ISSN: 0003-2697; 1096-0309; 1096-0309
• DOI: 10.1016/j.ab.2024.115697

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. [Display omitted] •Primary UB-PCR uses dUTP instead of dTTP as substrate.•The single-primer single-cycle PCR only replicates a U-DNA with pnSSP site(s).•UNG treatment improves the specificity of UB-PCR.•Two rounds of UB-PCRs can generally produce a wanted walking result.