A pair of primers facing at the double-strand break site enables to detect NHEJ-mediated indel mutations at a 1-bp resolution.

ABSTRACT: The introduction of small insertion/deletion (indel) mutations in the coding region of genes by the site-specific nucleases such as Cas9 allows researchers to obtain frameshift null mutants. Technically simple and costly reasonable genotyping methods are awaited to efficiently screen the frameshift null mutant candidates. Here, we developed a simple genotyping method called DST-PCR (Double-strand break Site-Targeted PCR) using "face-to-face" primers where the 3' ends of forward and reverse primers face each other at the position between 3-bp and 4-bp upstream of the PAM sequence, which is generally the Cas9-mediated double-strand break site. Generated amplicons are directly subjected to TBE-High-Resolution PAGE, which contains a high concentration of bis-acrylamide, for mutant clones detec

SUBMITTER: Ijaz F 

PROVIDER: S-EPMC9270360 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature