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Separating PCR products generated from wild-type versus mutant alleles with small indels based on size is beyond the resolution capacity of regular agarose gel electrophoresis. To overcome this limitation, we developed a simple genotyping method that exploits the differential electrophoretic mobility of homoduplex versus heteroduplex DNA hybrids in high concentration agarose gels. First, the CRISPR target region is PCR amplified and homo- and hetero-duplexed amplicons formed during the last annealing cycle are separated by 4\u20136% agarose gel electrophoresis. WT\/mutant heteroduplexes migrate more slowly and are distinguished from WT or mutant homoduplexes. Heterozygous alleles are immediately identified as they produce two distinct bands, while homozygous wild-type or mutant alleles yield a single band. To discriminate the latter, equal amounts of PCR products of homozygous samples are mixed with wild-type control samples, subjected to one denaturation\/renaturation cycle and products are electrophoresed again. Samples from homozygous mutant alleles now produce two bands, while those from wild-type alleles yield single bands. This method is simple, fast and inexpensive and can identify indels &gt;2\u2009bp. in size in founder pups and genotype offspring in established transgenic mice colonies.<\/jats:p>","DOI":"10.1038\/s41598-019-39950-4","type":"journal-article","created":{"date-parts":[[2019,3,14]],"date-time":"2019-03-14T12:06:06Z","timestamp":1552565166000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":48,"title":["A simple genotyping method to detect small CRISPR-Cas9 induced indels by agarose gel electrophoresis"],"prefix":"10.1038","volume":"9","author":[{"given":"Debanjan","family":"Bhattacharya","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2444-7707","authenticated-orcid":false,"given":"Erwin G.","family":"Van Meir","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2019,3,14]]},"reference":[{"key":"39950_CR1","doi-asserted-by":"publisher","first-page":"347","DOI":"10.1038\/nbt.2842","volume":"32","author":"JD Sander","year":"2014","unstructured":"Sander, J. 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