PRIMA: a rapid and cost-effective genotyping method to detect single-nucleotide differences using probe-induced heteroduplexes

Abstract

Abstract Targeted mutagenesis by programmable site-specific nucleases like CRISPR typically produce 1-base pair (bp) insertion or deletion (indel) mutations. Although several methods have been developed to detect such 1-bp indels, each method has pros and cons in terms of cost and/or resolution. Heteroduplex mobility assay (HMA) is a traditional technique detecting small base pair differences but it has a limited resolution of mutation size and the band patterns are often complex. Here, we developed a new method called PRIMA (Probe-Induced HMA) using a short single-stranded DNA molecule as a probe in HMA. By utilizing a 40-mer probe containing a 5-nucleotide deletion, we assessed the mobility of a heteroduplex with a target DNA fragment from a plant, bacterium, and human. This method allowed us to detect a 1-bp indel mutation consistently. We also showed that SNPs can be detected using PRIMA. PRIMA provides a rapid and cost-effective solution for the genotyping.