A simple genotyping method to detect small CRISPR-Cas9 induced indels by agarose gel electrophoresis View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2019-12

AUTHORS

Debanjan Bhattacharya, Erwin G. Van Meir

ABSTRACT

CRISPR gene editing creates indels in targeted genes that are detected by genotyping. 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 4-6% 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 >2 bp. in size in founder pups and genotype offspring in established transgenic mice colonies. More... »

PAGES

4437

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/s41598-019-39950-4

DOI

http://dx.doi.org/10.1038/s41598-019-39950-4

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1112769082

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/30872606


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