{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,20]],"date-time":"2026-02-20T06:24:00Z","timestamp":1771568640744,"version":"3.50.1"},"reference-count":42,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2025,8,1]],"date-time":"2025-08-01T00:00:00Z","timestamp":1754006400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Open Fund Project of the Biomedical Research Center","award":["BRC-KF202302"],"award-info":[{"award-number":["BRC-KF202302"]}]},{"name":"Open Fund Project of the Biomedical Research Center","award":["31920250026"],"award-info":[{"award-number":["31920250026"]}]},{"name":"Open Fund Project of the Biomedical Research Center","award":["20225BCJ22023"],"award-info":[{"award-number":["20225BCJ22023"]}]},{"name":"Fundamental Research Funds for the Central Universities of Northwest Minzu University","award":["BRC-KF202302"],"award-info":[{"award-number":["BRC-KF202302"]}]},{"name":"Fundamental Research Funds for the Central Universities of Northwest Minzu University","award":["31920250026"],"award-info":[{"award-number":["31920250026"]}]},{"name":"Fundamental Research Funds for the Central Universities of Northwest Minzu University","award":["20225BCJ22023"],"award-info":[{"award-number":["20225BCJ22023"]}]},{"name":"Major Discipline Academic and Technical Leaders Training Program of Jiangxi Province","award":["BRC-KF202302"],"award-info":[{"award-number":["BRC-KF202302"]}]},{"name":"Major Discipline Academic and Technical Leaders Training Program of Jiangxi Province","award":["31920250026"],"award-info":[{"award-number":["31920250026"]}]},{"name":"Major Discipline Academic and Technical Leaders Training Program of Jiangxi Province","award":["20225BCJ22023"],"award-info":[{"award-number":["20225BCJ22023"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["CIMB"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/cimb47080602","type":"journal-article","created":{"date-parts":[[2025,8,5]],"date-time":"2025-08-05T08:46:55Z","timestamp":1754383615000},"page":"602","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Center Degenerated Walking-Primer PCR: A Novel and Universal Genome-Walking Method"],"prefix":"10.3390","volume":"47","author":[{"given":"Dandan","family":"Gao","sequence":"first","affiliation":[{"name":"Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhenkang","family":"Pan","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Food Science and Resources, Nanchang University, Nanchang 330047, China"},{"name":"Sino-German Joint Research Institute, Nanchang University, Nanchang 330047, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hao","family":"Pan","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Food Science and Resources, Nanchang University, Nanchang 330047, China"},{"name":"Sino-German Joint Research Institute, Nanchang University, Nanchang 330047, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yinwei","family":"Gu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Food Science and Resources, Nanchang University, Nanchang 330047, China"},{"name":"Sino-German Joint Research Institute, Nanchang University, Nanchang 330047, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5001-4051","authenticated-orcid":false,"given":"Haixing","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Food Science and Resources, Nanchang University, Nanchang 330047, China"},{"name":"Sino-German Joint Research Institute, Nanchang University, Nanchang 330047, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Yik, M.H.Y., Lo, Y.T., Lin, X., Sun, W., Chan, T.F., and Shaw, P.C. 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