A Simple, Robust, and Cost-effective Method for Genotyping Small-scale Mutations

Abstract

Background and objectives: Genotyping is crucial for studying gene functions in animals and detecting genetic variants in humans. Traditional methods using agarose or polyacrylamide gel electrophoresis focus on detecting large-size differences (20–2,000 bp) between wild-type and mutant genes. While endonuclease digestion can identify heterozygous mutations, it fails to distinguish wild-type genes from homozygous mutants of similar size. This study aimed to develop a novel, simple, and reliable genotyping method for animals or cells following genetic modifications. Methods: We introduced an improved genotyping method utilizing 2% agarose gel electrophoresis after T7E1 or Surveyor endonuclease digestion to initially separate heterozygous mutations from wild-type and homozygous mutations. By adding a wild-type PCR product to potentially homozygous samples, forming heteroduplexes, we differentiated wild-type from homozygous mutations with nearly identical sizes or single base pair substitutions without relying on Sanger sequencing. Results: This method was validated in genotyping zebrafish mutants with 2-8 bp deletions or insertions and mouse mutants with 1-or 8-bp substitutions. Agarose gel clearly distinguished wild-type, heterozygous, and homozygous mutations ranging from 1–8 bp. Sanger sequencing confirmed the accuracy of our genotyping results. Conclusions: Our novel and improved genotyping method offers a rapid, economical approach for genotyping small deletions or single base pair substitutions. This technique has broad applications in clinical and research laboratories, especially in the era of gene editing and for detecting naturally occurring mutations.