A Simple CELI Endonuclease-Based Protocol for Genotyping both SNPs and InDels

Modern genetic analyses rely on efficient genotyping of single-nucleotide polymorphisms (SNP) or insertion/deletion length polymorphisms (InDel) in genomes. Methods available to genotype these polymorphisms include sequencing, cleaved amplified polymorphic sequence, high-resolution DNA melting, and microarray analyses, which are all rather tedious or expensive to set up for daily use. Here, we report a simplified label-free CELI endonuclease (CELI)-based protocol that enables us to detect both SNPs and InDels for fragment lengths between 500 and 6 kb. PCR-amplified target DNA fragments were annealed, cleaved by CELI, and analyzed either cost-effectively by agarose gel electrophoresis or automatically by capillary electrophoresis. The optimal amplification sizes, potential blind ends, and the maximum pooling capacities were examined for both electrophoresis protocols. We believe that the CELI-based genotyping protocol can be used in the detection of mutations, marker-assisted breeding, map-based cloning, and genome-wide association studies.