A Quick and Efficient Method for Detecting Single-Nucleotide Mutations: “PRIMA” Detection of Genome-Edited Mutants Using MultiNA™

Introduction

We developed a method that enables the detection of 1-base pair (bp) insertions/deletions (indels). This method is advantageous compared with existing methods such as the heteroduplex mobility assay (HMA). Although the HMA is an efficient method for genotyping mutants with small indels, it is difficult to distinguish sequences with 1-bp indels using the HMA. The probe-induced heteroduplex mobility assay (PRIMA) has made it possible to identify 1-bp differences with the use of 40-bp single-stranded DNA sequences with five deleted bases as a co-migrating probe. To genotype F2 individuals, the HMA requires two analysis runs to distinguish three genotypes (i.e., wild-type homozygous, heterozygous, and mutant homozygous); however, the PRIMA can distinguish three genotypes in a single run. It can also be used to detect single- nucleotide polymorphisms (SNPs). We succeeded in distinguishing SNPs with the PRIMA using a Shimadzu MultiNA automated microchip electrophoresis system.

December 25, 2022 GMT