| With the success of Human Genome Project (HGP), more and more research interests in the field of life science have been turned to the function, expression and regulation of genes, the relationship among genes and environments. One of the fruits of the Human Genome Project is the research of single nucleotide polymorphism (SNP). As the third generation of genetic markers, SNPs are so highly abundant that can be use in mapping or cloning of disease-related genes, and association studies, what's more, may play a very important role in diagnosis, prevention, and therapy of complex traits.That a large number of individuals must be genotyped requires an efficient, high-throughput and low-cost SNP genotyping method. To date, there are mainly two microarray-based methods for genotyping. One is arraying thousands of short oligonucleotides to glass slide for detection of all possible SNPs loci in target DNA. The other approach involves arraying amplified PCR products to glass slides to detect one or many SNPs loci for a large of samples. The aim of the present study was to develope a novel method for genotyping of functional SNPs in hundreds of individuals based on 3-D gel DNA microarray. The main contents are as follows:1. Optimization of 2-D DNA microarray for high-throughput SNP genotypingThree different chemistry-modified (APTES, glutaraldehyde, and poly-L-Lysine) glass slides, and different length of PCR products were evaluated based on dual-color fluorescence hybridization. The results showed that about 200bp products immobilized on aldehyde-coated slides exhibited more stable signal intensity due to the steady schiff bond, and high signal-noisy ratio. However, the large amounts of products requirement and low efficiency of hybridization exposed the drawback of this kind of 2-D microarray.2. Preparation and optimization of 3-D gel microarray for high-throughput SNP genotypingCompared with traditional 2-D microarray, 3-D gel microarray could immobilize much more DNA, and provided a homogenous-like reaction circumstance and a stable support with low fluorescent background. The acryl-modified slides spooted with prepolymer were transfered in a vacuous chamber filled with TEMED. Incubated for one hour, polyacrylamide gel-based microarray for SNP genotyping was prepared by copolymerizing parallel amplified acrylamide-modified PCR products with acrylamide monomers. After dual-color fluorescence hybridization, non-specific binding labeled targets were removed by applying an electric field. Furthermore, optimization about polyacrylamide concentration, denature method of sample microarray, length of probes, condition of electrophoresis were carried on. The assay format based on gel-immobilization achieves a high probe density and has the ability to use unpurified PCR products for array formation. Meanwhile, universal tag probes were proposed to decrease the cost greatly.3. Association analysis of SNPs with risk of coronary heart disease (CHD) based on polyacrylamide gel microarrayMethod of functional SNPs selection based on bioinformatics was proposed. 7 SNPs of 6 genes (OLR1, MMP-1, MMP-13, MMP-11, MMP-27, MMP-L1) were selected. 238 CHD patients and 233 healthy... |
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