Laboratory of Molecular Neuro-Oncology, Departments of Neurosurgery and Hematology & Medical Oncology, Emory University School of Medicine, Atlanta, GA, 30322, USA.
Winship Cancer Institute, Emory University, Atlanta, GA, 30322, USA.
Sci Rep. 2019 Mar 14;9(1):4437. doi: 10.1038/s41598-019-39950-4.
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.
CRISPR 基因编辑会在靶基因中产生插入缺失（indels），这些插入缺失可以通过基因分型检测到。基于大小，用常规琼脂糖凝胶电泳分离具有小插入缺失的野生型和突变型等位基因的 PCR 产物超出了分辨率能力。为了克服这一限制，我们开发了一种简单的基因分型方法，该方法利用了高浓度琼脂糖凝胶中同源双链体与异源双链体 DNA 杂交体的不同电泳迁移率。首先，对 CRISPR 靶区域进行 PCR 扩增，在最后一个退火循环中形成同源和异源双链体扩增子，并通过 4-6%琼脂糖凝胶电泳进行分离。WT/突变异源双链体迁移速度较慢，与 WT 或突变型同源双链体区分开来。杂合等位基因可以立即识别出来，因为它们产生两条不同的带，而纯合野生型或突变型等位基因则产生一条带。为了区分后者，将纯合样本的等量 PCR 产物与野生型对照样本混合，进行一次变性/复性循环，然后再次电泳。来自纯合突变等位基因的样本现在产生两条带，而来自野生型等位基因的样本则产生单条带。这种方法简单、快速且廉价，可以鉴定大小大于 2bp 的插入缺失。在建立的转基因小鼠品系中，用于鉴定 founder 幼鼠和基因型后代。
