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 （ME...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 Cy3-1abeled 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 genotvping analysis for these four SNP loci was carried out successfully simultaneously by this method.展开更多
基金financially supported by the National Key Program for Developing Basic Research(No.2010CB933903)the Chinese National Key Project of Science and Technology(No.2013ZX10004103-002)+2 种基金the National Youth Science Foundation of China(No.61301043)the NSFC(Nos.61271056,61471168,61201100 and 61527806)the Economical Forest Cultivation and Utilization of 2011 Collaborative Innovation Center in Hunan Province[No.(2013)448]
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 Cy3-1abeled 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 genotvping analysis for these four SNP loci was carried out successfully simultaneously by this method.
