Multiple genotyping based on multiplex PCR and microarray

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• 作者：Xian-Bo Mou^a ; Zeeshan Ali^a ; Bo Li^a ; Tao-Tao Li^a ; Huan Yi^a ; Hong-Ming Dong^b ; Nong-Yue He^a ; ^b ; ^{nyhe1958@163.com" class="auth_mail" title="E-mail the corresponding author} ; Yan Deng^b ; ^{hndengyan@126.com" class="auth_mail" title="E-mail the corresponding author} ; Xin Zeng^c ; ^{august555482@126.com" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Magnetic beads ; Multiplex PCR ; Microarray ; Multiple genotyping ; Gastric cancer
• 刊名：Chinese Chemical Letters
• 出版年：2016
• 出版时间：November 2016
• 年：2016
• 卷：27
• 期：11
• 页码：1661-1665
• 全文大小：1086 K

文摘

The genetic variability has obtained more and more attention in the process of diagnosis and treatment of tumors. Herein, we have described a multiple genotyping method based on magnetic enrichment-multiplex PCR (MEM-PCR) and microarray technology. Monodisperse magnetic beads were fabricated and modified with streptavidin. Four loci on two genes (M235T and A-6G loci on AGT gene, A1298C and C677T loci on MTHFR gene) were selected to study single nucleotide polymorphisms (SNP). Target sequences of these SNP loci were amplified using Cy3-labeled primers through multiplex PCR in one tube after the templates were enriched and purified by functional magnetic beads (MB). Four pairs of NH₂-labeled probes, corresponding to each locus, were fixed on CHO-modified glass slide by covalent binding. Hybridization between target sequences and probes was performed under suitable conditions. The spotting locations on microarray and the ratio of fluorescence intensity, produced by different loci, were used to distinguish the SNP genotypes. Finally, three of gastric cancer samples were collected and genotyping analysis for these four SNP loci was carried out successfully simultaneously by this method.