A novel and cost-efficient allele-specific PCR method for multiple SNP genotyping in a single run

Cost-effective methods for DNA genotyping were needed because single nucleotide polymorphisms (SNPs) were essential biomarkers associated with many diseases. Allele-specific PCR (AS-PCR) has the advantages of mature instruments and high sensitivity. But conventional AS-PCR needs to multiply the number of reactions or primers for multiple targets, which complicates the operation and increases the cost. Herein, we proposed a novel AS-PCR method for multiple SNP genotyping in a single run. Wild-type allele-specific primer (WT primer) was designed for each target gene. The sample and WT primers only needed to undergo multiplexed AS-PCR once simultaneously. After AS-PCR, the concentration of remaining primers varied among the samples of each genotype combination, due to the different matching performance between template and WT primers. The remaining primers then triggered multiplexed molecular beacon-rolling circle amplification, and the molecular beacons labelled with different fluorescent dyes corresponded to different targets. The fluorescence ratios of the sample to the positive control were used as the genotyping indexes. This method was able to detect samples with concentrations as low as 10 fM. We successfully applied the method to the multiple genotyping of 23 hair root samples for ADH1B and ALDH2 genes, obtaining completely consistent results with sequencing. The reagent cost was 0.6 dollar for one sample, showing a good cost performance. This proposed approach had a great application prospect in simultaneously rapid and accurate genotyping of multi-SNPs, and provided a new method for personalized health management.