High-throughput genotyping by coupling adapter-ligation mediated allele-specific amplification with microplate array...

Recently, we have developed a method of adapter-ligation mediated allele-specific amplification (ALM-ASA) for simultaneously typing multiple single nucleotide polymorphisms (SNPs) at a low cost. We usually use agarose gel-electrophoresis for analyzing PCR products. As the processes of sampling and PCR can be carried out at a format of 96-well or 384-well, the throughput-bottleneck of whole process of ALM-ASA is only the agarose gel-electrophoresis. Here we improved the typing throughput of ALM-ASA by using a microplate array parallel gel electrophoresis (MAPGE) system, with which 96 amplicons can be detected at a time. By coupling with multiplexed preamplification, seven SNPs distributed on four different human genes (IL1A (549C>T), 1L1B (794C>T and 5277C>T), IL10 (2940G>A, 3203C>T, and 3430C>A), and TNFA (1431G>A)) were successfully typed. The optimization of allele-specific primers in ALM-ASA was performed by the software of "SNiPdesigner" which was designed especially for ALM-ASA. We also demonstrated that the specificity of ALM-ASA assay for SNP typing is superior to that of amplification refractory mutation system (ARMS).