High-throughput SNP Genotyping With PCR Amplification On Magnetic Nanoparticles

Single nucleotide polymorphisms (SNPs) are the most frequent forms of sequence variations in the human genome and the studies of them may offer the key to understand genetic differences between individuals and disease states, and eventually improve medical treatments by allowing the prediction of genetically related disease risk and drug response. Therefore, it is very necessary to establish a rapid, accurate and suitable for clinic method for the high-throughput genotyping of SNPs. Over the past several years, numerous high-throughput and cost-effective methods to discover and measure SNPs have been developed as attempts to achieve these goals. In fact, however, when dealing with a large number of samples, the necessary steps of concentration and purification for target DNA usually make most of the methods time-consuming and not rigid enough for the clinical applications.With the rapid advancing of nanotechnology in life science, nano-materials have become a focus of interest in various fields of biological and biomedical application. As one of the most important nanostructured materials, magnetic nanoparticles (MNPs) have been widely applied in cell separation, immunoassays, the immobilization of protein and enzyme and DNA detection due to their higher dispersion capability in aqueous solution and their higher separation efficiency in magnetic field.Herein, combining the easy separation characteristic of MNPs and the high-throughput"read-out"characteristic of biochips, this thesis described a novel high-throughput SNP genotyping method to detect the SNPs of the methylenetetrahydrofolate reductase (MTHFR) gene containing the C677T SNP locus using magnetic nanoparticles as PCR primers carriers and allele-specific probes labeled dual-color hybridization. 126 samples of MTHFR gene containing the C677T SNP locus were detected by using this method. The results indicated that when one biotin-labeled primer was bounded to the surface of MNPs modified streptavidin (SA-MNPs), PCR could proceed successfully and its amplified fragment was 213bp. The PCR products amplified on MNPs were hybridized with a pair of dual-color fluorescence (Cy3, Cy5) probes at the temperature of 38℃, and then genotype of each sample can be simultaneously identified by scanning the microarray printed with the denatured fluorescent probes on an unmodified glass slide. The genotyping results were additionally validated by sequencing.To reduce the cost of genotyping, on the base of MNPs-PCR method as described above, wild and mutant tagged probes and a pair of dual-color (Cy3, Cy5) universal detectors were designed to determine SNPs. The C677T and M235T loci of 96 different samples were detected by this method and the reactive signal was obtained by scanning the denatured fluorescence probes spotted on the slide. The genotyping results were consistent with the results of sequencing. To validate the accurate of this method applied in automated workstation system, the M235T locus of 96 samples were detected using this method. The hybridization, denaturation and washing steps were performed in a 96-well plate of the workstation automaticly, which made the detection procedure become much simplier and faster than the previously reported genotyping methods, and the expected scores and good discrimination were obtained when automated workstation was used. Without any purification and concentration of PCR products, the two kinds of high-throughput genotyping methods described above should be simple, high-throughput and high accurate. All the procedure could be accomplished in 96- or 384-well plate, which will make a fully automated system for genotyping or DNA detection more easily constructable.