Single-tube genotyping for small insertion/deletion mutations: Simultaneous identification of wild type, mutant and heterozygous alleles

Abstract

Current methods of genotyping small insertion/deletion (indel) mutations are costly, laborious, and can be unreliable. To address this, we have developed a method for small indel genotyping in a single polymerase chain reaction, with wild-type, heterozygous and mutant alleles distinguishable by band pattern in routine agarose gel electrophoresis. We demonstrate this method with multiple genes to distinguish 10 bp, 4 bp and even 1 bp deletions from the wild type. Through systematic testing of numerous primer designs, we also propose guidelines for genotyping small indel mutations. Our method provides a convenient approach to genotyping small indels derived from clustered regularly interspaced short palindromic repeats-mediated gene editing, N-ethyl-N-nitrosourea induced mutagenesis or diagnosis of naturally occurring polymorphisms/mutations.