Molecular Genetics, Leibniz Institute on Aging - Fritz Lipmann Institute, 07745 Jena, Germany
Molecular Genetics, Leibniz Institute on Aging - Fritz Lipmann Institute, 07745 Jena, Germany.
Cold Spring Harb Protoc. 2023 Aug 1;2023(8):107744. doi: 10.1101/pdb.prot107744.
CRISPR-Cas9 has eased the induction of sequence-specific mutations and has therefore become a powerful tool to generate mutant lines for studying the role of specific genes. The cellular repair of Cas9-induced double-stranded DNA breaks by the error-prone nonhomologous end-joining pathway can result in various indel mutations. Having identified and chosen a specific mutation in a target gene, the establishment of a respective mutant line requires a feasible and precise method to differentiate the genotypes of the offspring. Here, we provide a protocol that allows genotyping of large numbers of embryos, larvae, and adults harboring a previously identified indel or point mutation in a short time via high-resolution melt analysis (HRMA).
CRISPR-Cas9 简化了序列特异性突变的诱导，因此成为生成用于研究特定基因作用的突变系的强大工具。由易错的非同源末端连接途径修复 Cas9 诱导的双链 DNA 断裂可导致各种缺失插入突变。在鉴定和选择靶基因中的特定突变后，建立相应的突变系需要一种可行且精确的方法来区分后代的基因型。在这里，我们提供了一种通过高分辨率熔解分析（HRMA）在短时间内对大量携带先前鉴定的缺失或点突变的胚胎、幼虫和成虫进行基因分型的方案。
