Application Of SNP Analysis By Pfu High-fidelity DNA Polymerase With Double Phosphorothioate Modified Allele Specific Primers In Direct Genotyping From Whole Blood

Using the high fidelity pfu DNA polymerase with phosphorothioate modified allele specific primers, established accurate and sensitive method for SNP genotyping. This study developed a rapid, highly specific genotyping method to genotype warfarin dosing related SNPs in VKORC1 and CYP2C9 genes. This study further developped a method to amplify DNA direct from whole blood with pfu DNA polymerase and found that dUTP can be used in whole bood PCR with UNG to eliminated PCR product carry-over contamination. Combined with the whold blood PCR method, and specific genotyping method by Pfu DNA polymerase and phosphorathioate primers, direct whole blood genotyping method was used to screen genotypes from blood samples for cyp2c9 amd vkorc1. The resutls were completely consistent with that of direct sequencing. Finally, this study established the highly specific, rapid genotype testing technology using whole blood, and then successfully applicated in clinical genetic testing for warfarin dosing.Methods:Based on literature information, identified that the Han population in mainland China the dose of warfarin individual differences is mainly caused by in the two related genes, VKORC1 and CYP2C9. Identified the SNP positions sequences based on the sequences of these two genes in NCBI Gene database. Cloning DNA fragments within the SNP positions sequences, and then obtained mutants by site-directed mutagenesis and produced the recombinant plasmid as a template for research. Designing the serveral primers to detected SNP, By doing extensive and repeated the experiments to test the specificity of primers and PCR conditions, the best primers will be applied in the SNP analysis. Optimization of PCR reaction and amplification conditions, also added PCR products anti-pollution system, to achieve high specific rapid genotype testing with whole blood.Results:We have identified the gene sequence and the SNP location; Cloned the DNA fragments within the SNP positions sequences, and obtained mutants by site-directed mutagenesis, constructed recombinant plasmids containing the wild-type and mutant templets; Through a large number and repeated experiments confirmed that Salmon DNA playing a very important role in plasmid detection system. Adding 50ng of Salmon DNA to 0pg/25μl plasmid detection system can achieve accurate and realistic detecttion the effect of SNP. It showed unique advantages in SNP analysis with pfu high-fidelity DNA polymerase with phosphorothioate modified allele specific primers for its accuracy. We optimized the best detection system contains 20ng genomic DNA templates and 0.75U Pfu polymerase. We further found that by using Touchdowm PCR method can increase specificity of PCR to avoid the production of non-specific PCR products. We have successfully established a PCR anti-pollution system to avoid product contamination. In combination of pollution prevention system, high fiedelity pfu polymerase and modified PCR polmers, we have established a high specific and rapid whole blood mutation detection. With this method, genotype analysis can be down within 3 hours. We have found that 8% whole blood and 3 mM magnesium could achieve high efficiency PCR amplification with high specificity. The types of anti-cogluatants had no effect on either PCR efficiency or specificity.Conclusions:In the plasmid detection system, addition of Salmon DNA significantly improved the efficiency and accuracy for optimization of genotype-specific primers. We have successfully established a method with the human genome DNA as a template for developing accurate and sensitive SNP genotyping method. We have successfully established a method for rapid SNP genotyping with the whole blood. A SNP genotyping kit for warfarin dosing has been developed based this methodology and is now in the process of commercialization.