{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2024,8,29]],"date-time":"2024-08-29T00:29:43Z","timestamp":1724891383178},"reference-count":17,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2024,8,28]],"date-time":"2024-08-28T00:00:00Z","timestamp":1724803200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2024,8,28]],"date-time":"2024-08-28T00:00:00Z","timestamp":1724803200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"name":"Universit\u00e4tsklinikum Freiburg"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>PCR is tolerant to single nucleotide mismatches. Therefore, genotyping of point mutations by PCR requires special conditions for the amplification of allele-specific PCR fragments. MS-PCR (mutagenically separated PCR) is an improved version of ARMS (amplification refractory mutation system) in which additional nucleotide mismatches near the mutation site are used to separate the wt fragments from the mutant fragments in a single-tube PCR. In the originally described procedure, the resulting fragments are resolved on agarose gels according to differences in size introduced by different lengths of the allele-specific primers. In order to evaluate the PCR fragments by melting curve analysis, we enlarged the difference in the melting temperatures of the fragments of the two alleles by increasing the GC content of the longer allele-specific primer resulting in a higher melting temperature of the corresponding fragment. Using the murine retinal degeneration mutations rd1 and rd8 as an example, we show that such primers result in an easy to handle genotyping procedure: qPCR followed by melting curve analysis. In summary, MS-PCR is a simple and easy-to-use method for detecting single nucleotide variants.<\/jats:p>","DOI":"10.1038\/s41598-024-70949-8","type":"journal-article","created":{"date-parts":[[2024,8,28]],"date-time":"2024-08-28T04:02:55Z","timestamp":1724817775000},"update-policy":"http:\/\/dx.doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Genotyping single point mutations in rd1 and rd8 mice using melting curve analysis of qPCR fragments"],"prefix":"10.1038","volume":"14","author":[{"given":"Melanie E.","family":"Schw\u00e4mmle","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Felicitas","family":"Bucher","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"G\u00fcnther","family":"Schlunck","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gottfried","family":"Martin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2024,8,28]]},"reference":[{"key":"70949_CR1","first-page":"63","volume":"27","author":"CDS Mamotte","year":"2006","unstructured":"Mamotte, C. D. S. Genotyping of single nucleotide substitutions. Clin. Biochem. Rev. 27, 63\u201375 (2006).","journal-title":"Clin. Biochem. Rev."},{"key":"70949_CR2","doi-asserted-by":"publisher","first-page":"2316","DOI":"10.3390\/ijms18112316","volume":"18","author":"M S\u0142omka","year":"2017","unstructured":"S\u0142omka, M., Sobalska-Kwapis, M., Wachulec, M., Bartosz, G. & Strapagiel, D. High resolution melting (HRM) for high-throughput genotyping-limitations and caveats in practical case studies. Int. J. Mol. Sci. 18, 2316 (2017).","journal-title":"Int. J. Mol. Sci."},{"key":"70949_CR3","first-page":"293","volume":"29","author":"M Tamburro","year":"2017","unstructured":"Tamburro, M. & Ripabelli, G. High resolution melting as a rapid, reliable, accurate and cost-effective emerging tool for genotyping pathogenic bacteria and enhancing molecular epidemiological surveillance: A comprehensive review of the literature. Ann. Ig. Med. Prev. E Comunita 29, 293\u2013316 (2017).","journal-title":"Ann. Ig. Med. Prev. E Comunita"},{"key":"70949_CR4","doi-asserted-by":"publisher","first-page":"2503","DOI":"10.1093\/nar\/17.7.2503","volume":"17","author":"CR Newton","year":"1989","unstructured":"Newton, C. R. et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 17, 2503\u20132516 (1989).","journal-title":"Nucleic Acids Res."},{"key":"70949_CR5","doi-asserted-by":"publisher","DOI":"10.1002\/0471142905.hg0908s07","author":"S Little","year":"1995","unstructured":"Little, S. Amplification-refractory mutation system (ARMS) analysis of point mutations. Curr. Protoc. Hum. Genet. https:\/\/doi.org\/10.1002\/0471142905.hg0908s07 (1995).","journal-title":"Curr. Protoc. Hum. Genet."},{"key":"70949_CR6","doi-asserted-by":"publisher","first-page":"e32866","DOI":"10.1371\/journal.pone.0032866","volume":"7","author":"DN Birdsell","year":"2012","unstructured":"Birdsell, D. N. et al. Melt analysis of mismatch amplification mutation assays (melt-MAMA): A functional study of a cost-effective SNP genotyping assay in bacterial models. PLoS\u00a0One 7, e32866 (2012).","journal-title":"PLoS\u00a0One"},{"key":"70949_CR7","doi-asserted-by":"publisher","first-page":"3623","DOI":"10.1093\/nar\/21.16.3623","volume":"21","author":"S Rust","year":"1993","unstructured":"Rust, S., Funke, H. & Assmann, G. Mutagenically separated PCR (MS-PCR): A highly specific one step procedure for easy mutation detection. Nucleic Acids Res. 21, 3623\u20133629 (1993).","journal-title":"Nucleic Acids Res."},{"key":"70949_CR8","doi-asserted-by":"publisher","first-page":"1540","DOI":"10.1093\/clinchem\/46.10.1540","volume":"46","author":"GG Donohoe","year":"2000","unstructured":"Donohoe, G. G., Laaksonen, M., Pulkki, K., R\u00f6nnemaa, T. & Kairisto, V. Rapid single-tube screening of the C282Y hemochromatosis mutation by real-time multiplex allele-specific PCR without fluorescent probes. Clin. Chem. 46, 1540\u20131547 (2000).","journal-title":"Clin. Chem."},{"key":"70949_CR9","first-page":"447","volume":"69","author":"A Dandana","year":"2011","unstructured":"Dandana, A. et al. Real time PCR for fast detection of the angiotensinogen polymorphisms. Ann. Biol. Clin. 69, 447\u2013451 (2011).","journal-title":"Ann. Biol. Clin."},{"key":"70949_CR10","doi-asserted-by":"publisher","first-page":"8322","DOI":"10.1073\/pnas.88.19.8322","volume":"88","author":"SJ Pittler","year":"1991","unstructured":"Pittler, S. J. & Baehr, W. Identification of a nonsense mutation in the rod photoreceptor cGMP phosphodiesterase beta-subunit gene of the rd mouse. Proc. Natl. Acad. Sci. U. S. A. 88, 8322\u20138326 (1991).","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"70949_CR11","first-page":"2529","volume":"39","author":"W Yan","year":"1998","unstructured":"Yan, W., Lewin, A. & Hauswirth, W. Selective degradation of nonsense beta-phosphodiesterase mRNA in the heterozygous rd mouse. Investig. Ophthalmol. Vis. Sci. 39, 2529\u20132536 (1998).","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"70949_CR12","doi-asserted-by":"publisher","first-page":"2179","DOI":"10.1093\/hmg\/ddg232","volume":"12","author":"AK Mehalow","year":"2003","unstructured":"Mehalow, A. K. et al. CRB1 is essential for external limiting membrane integrity and photoreceptor morphogenesis in the mammalian retina. Hum. Mol. Genet. 12, 2179\u20132189 (2003).","journal-title":"Hum. Mol. Genet."},{"key":"70949_CR13","doi-asserted-by":"publisher","first-page":"2921","DOI":"10.1167\/iovs.12-9662","volume":"53","author":"MJ Mattapallil","year":"2012","unstructured":"Mattapallil, M. J. et al. The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL\/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes. Investig. Ophthalmol. Vis. Sci. 53, 2921\u20132927 (2012).","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"70949_CR14","doi-asserted-by":"publisher","first-page":"275","DOI":"10.1186\/1471-2164-8-275","volume":"8","author":"P Wangkumhang","year":"2007","unstructured":"Wangkumhang, P. et al. WASP: A web-based allele-specific PCR assay designing tool for detecting SNPs and mutations. BMC Genom. 8, 275 (2007).","journal-title":"BMC Genom."},{"key":"70949_CR15","doi-asserted-by":"publisher","first-page":"1068","DOI":"10.2144\/03345dd03","volume":"34","author":"AC Papp","year":"2003","unstructured":"Papp, A. C., Pinsonneault, J. K., Cooke, G. & Sad\u00e9e, W. Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves. BioTechniques 34, 1068\u20131072 (2003).","journal-title":"BioTechniques"},{"key":"70949_CR16","doi-asserted-by":"publisher","first-page":"773956","DOI":"10.3389\/fmolb.2022.773956","volume":"9","author":"R Kalendar","year":"2022","unstructured":"Kalendar, R., Shustov, A. V., Akhmetollayev, I. & Kairov, U. Designing allele-specific competitive-extension PCR-based assays for high-throughput genotyping and gene characterization. Front. Mol. Biosci. 9, 773956 (2022).","journal-title":"Front. Mol. Biosci."},{"key":"70949_CR17","doi-asserted-by":"publisher","first-page":"e0217222","DOI":"10.1371\/journal.pone.0217222","volume":"14","author":"H Ayalew","year":"2019","unstructured":"Ayalew, H. et al. Comparison of TaqMan, KASP and rhAmp SNP genotyping platforms in hexaploid wheat. PLoS\u00a0One 14, e0217222 (2019).","journal-title":"PLoS\u00a0One"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-024-70949-8.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-024-70949-8","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-024-70949-8.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,8,28]],"date-time":"2024-08-28T04:09:55Z","timestamp":1724818195000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-024-70949-8"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,8,28]]},"references-count":17,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2024,12]]}},"alternative-id":["70949"],"URL":"https:\/\/doi.org\/10.1038\/s41598-024-70949-8","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,8,28]]},"assertion":[{"value":"22 May 2024","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"22 August 2024","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"28 August 2024","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"19909"}}