High-throughput SNP genotyping method with PCR on magnetic nanoparticles

Single nucleotide polymorphisms (SNPs) assay is important for elucidating the genetics of individual differences in drug response and disease susceptibility. This report described a novel high-throughput SNP genotyping method using magnetic nanoparticles as PCR primers carriers. PCR products were directly amplified on MNPs and interrogated by hybridization with a pair of dual-color fluorescence(Cy3, Cy5) probes to determine SNP, 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. As the results, the homozygous wild type, homozygous mutant type and heterozygote type yield strongly green, red and yellow fluorescent signals, respectively. The methylenetetrahydrofolate reductase(MTHFR) gene C677T polymorphism of nine different samples was detected and their fluorescent signals of the nine samples were quantified. The fluorescent ratios( match probe signal to mismatch probe signal) of homozygous samples were over 9.0, whereas heterozygous samples had ratios near 1.0. The genotyping results were additionally validated by sequencing. Without any purification and concentration of PCR products, the approach reported here should be a simple, sensitive, high-throughput and high accurate genotyping method.