Establishment And Pilot Application Of The High-Throughput Single Nucleotide Polymorphism Analysis System With TDI-FP Technology

TDI-FP is a novel technology used to analyze the genotype of Single Nucleotide Polymorphism (SNP). It was developed from Template-directed Dye-terminator Incorporation (TDI) with the Fluorescence Polarization (FP) as the detection tool. TDI-FP is a highly specific and sensitive, easy to handle, technology, and could be adapted to automatical and high-throughput SNP detection.In order to apply the TDI-FP technology to be used in basic studies of SNP and the detection of clinical disease-related SNP or point mutation in China, we established the high-throughput single nucleotide polymorphism detection systemInstitute of Genetic Diagnosis, Fourth Military Medical University ?6 --with TDI-FP and used it to detect the HBV YMDD motif mutations to determine the system's veracity and feasibility.We used the site-directed mutagensis plasmid of YMDD motif to optimize the PCR reaction system and digestion duration of the PCR Clean-Up reagent of the system. The optimal PCR reaction system was compromised of 5.l 10+ Reaction buffer, 31 MgCl2 (25mmol/L), 2.5l dNTPs (2.5mol/L), 0.5l each primer (25mol/L), 0.51 Taq polymerase (5U/l), 11 template and 37l ddH2O. The concentration of PCR product was ranged from 1.18ng/l to 7.08ng/l. The duration of the PCR clean-up reagent was at least 1.0h at 37 C to completely eliminate the redundant dNTPs and primers in the reaction system.Using this system, of the 35 serum samples analysed, the wild-type YMDD motif was detected in 26, the YVDD variant in 2, YMDD and YVDD coinfection in 2, YIDD variantin 2, YMDD and YIDD coinfection in 1. 2 samples were detected negative at the position 741. After sequenceing analysis, there was a G741-C mutation. The results of the high-throughput single nucleotide polymorphism analysis system were in perfect concordance with the direct sequencing results. It has a specificity of 100%. The system's sensitivity was directly related to the PCR reaction system, it was 92.1% (35/38).The TDI-FP technology used in this study is an accurate and specific mthod for genotyping SNPs. Since it is a rapid low-cost and high-throughput technique, TDI-FP has a high promising for large-scale screening of polymorphisms in clinical, pathogenetic microorganism and pharmacogenomic analysis. In order to analyze the results automatically, we used Visual Basic to write the codes of the software for high-throughput single nucleotide polymorphism analysis system of HBV DNA's mutations' based on Microsoft Excel books. The software wasInstitute of Genetic Diagnosis, Fourth Military Medical Universitydedicated to the automatical genotyping of the important point mutations in the four open reading frame of the HBV DNA. Users could analyze the genotypes according to the value of mP and scatter plot. The software was laid a sound foundation for the development of 'SNP Scorer' to analyze the human SNP automatically.