High-Throughput Genotyping by Coupling Adapter-Ligation Mediated Allele-Specific Amplification with Microplate Array Parallel Gel Electrophoresis |
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Authors: | Weipeng Wang Xiaodan Zhang and Guohua Zhou |
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Institution: | (1) Department of Pharmaceutical Analysis, Medical College, Soochow University, Suzhou, 215123, People’s Republic of China;(2) Huadong Research Institute for Medicine and Biotechnics, East Zhongshan Road 293, Nanjing, 210002, People’s Republic of China;(3) Medical School, Nanjing University, Nanjing, 210093, People’s Republic of China |
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Abstract: | 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). |
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