SNPs and single base pair (SBP) insertion/deletions (indels) are not only the most abundant genetic markers for genetic mapping and breeding selection, but also always occur in the mutants generated from chemical mutagenesis or CRISPR/Cas9-mediated genome editing. Most of the current SNP and SBP indel genotyping methods are time-consuming and/or require special equipment or reagents. Here, we describe an improved heteroduplex analysis method, named iHDA, that can readily discriminate SNP and SBP indel alleles with specially designed DNA probes that harbor a couple of nucleotides adjacent to the SNP site. By hybridizing with the same probe, SNP and SBP indel alleles form different heteroduplexes, differing in bulge size, which show different mobility on a polyacrylamide gel. Therefore, iHDA is an easy, fast and inexpensive method for SNP and SBP indel genotyping.
Keywords: CRISPR; genotyping; heteroduplex assay; iHDA; improved heteroduplex assay; single base pair insertion/deletion; single nucleotide polymorphism.