The CYP2C19 Gene Was Subjected To Double SNP Typing And TALEN-mediated Mouse P53 Knockout By HRM Technique

Part ⅠDuplex genotyping of CYP2C19*2and CYP2C19*3by high-resolution melting curve analysisClopidogrel is a widely used anti-platelet agent for the prevention of arterial thrombosis. It has been suggested that clopidogrel may be less effective in inhibiting platelet aggregation among patients who are carriers of CYP2C19*2and CYP2C19*3, two loss-of-function CYP2C19alleles, which are associated with reduced conversion of clopidogrel to its active metabolite. The objective of this research was to develop a simple and accurate method for genotyping of CYP2C19*2and CYP2C19*3simultaneously in one closed-tube using high-resolution melting curve (HRM) analysis. Two amplicons bracketing CYP2C19*2and CYP2C19*3gene variants were designed, and AT-or GC-rich5’tails were added to selected primers to ensure two different amplicons with non-overlapping melting curves.64random DNA samples were all fast and sensitively genotyped by HRM analysis. This method w(?) validated by DNA sequencing techniques, and genotypes obtained using the HRM approach perfectly matched the genotypes obtained by DNA sequencing techniques. Therefore, this HRM-based assay allows simple and accurate duplex genotyping of CYP2C19*2and CYP2C19*3simultaneously in one closed-tube. This method is expected to be applied in clinical laboratory to guide individual dosage design of Clopidogrel. Part Ⅱp53gene modification with TALEN technologyTAL effector nucleases (TALENs) are fusion proteins which are produced by combining the DNA recognition domain of transcription activator-like (TAL) effectors with the nuclease domain of FokI restriction enzyme. They can, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining or homologous recombination. As an important new tools for genome engineering, they are now widely applied in site-specific gene modification and mutation.The aim of this research was to modificate p53gene in living cells with TALEN technologies. We assembled TALEN constructs and the cutting efficiency tested in yeast was3.5%. We transfected NIH3T3cells with TALEN constructs and successfully detected gene mutations with HRM analysis. We co-transfected NIH3T3cells with TALEN constructs and p53donor DNA to observe the expression of GFP protein after homologous recombination. GFP expression and puromycin resistant cells were abtained with puromycin selection, the final concentration of which was8ug/ml. When the number of selected cells grew up, we confirmed the homologous recombination by PCR method and analysed the cell circle changes between normal cells and selected cells. The PCR confirmation shows that p53gene was correctly recombined. Cell circle analysis showed that the cell circle was blocked to a certain degree.All evidences above shows that TALENs can cleave DNA double strands intracellular efficiently and can be used in gene modification. This technology provides robust theoretical and technical basis for gene therapy and gene modification.