High-throughput genotyping by coupling adapter-ligation mediated allele-specific amplification with microplate array parallel gel electrophoresis.
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).
Wang
Weipeng
W
Department of Pharmaceutical Analysis, Medical College, Soochow University, Suzhou, 215123, People's Republic of China. wangweipeng@suda.edu.cn
Zhang
Xiaodan
X
Zhou
Guohua
G
D000465:Algorithms; D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012984:Software; D018511:Systems Integration
D004247:DNA
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