The Application Of Improved "Molecular Switch" Technology In Detecting The CYP2C19*2and CYP2C19*3

Objective: Clopidogrel belongs to the phenothiazine pyridine anti-platelet drugthat be widely used in cardiovascular and cerebrovascular diseases, but clinically thereare significant individual-differences in Clopidogrel metabolism. Studies have showsthat CYP2C19*2(681G>A) and CYP2C19*3(636G>A) nucleotide polymorphismsrelate to drug metabolism, if the two alleles could be detected accurately that will have agreat significance to clinical treatment for Clopidogrel. This study aims to establish ahigh-sensitivity and simply experiment technology that could be used for SNP screening,in the meanwhile that will be foundation for drug guidance of Clopidogrel.Methods:1Based on "molecular switch" technology for SNP genotyping, we designspecific primers that3′end phosphorthioational combination with high-fidelity PfuDNA polymerase which has3′â†'5′exonuclease proofreading activity, select DNAwhich the two alleles genotype already be known as templates to amplify to explore themost suitable PCR reaction condition for establishing the stable method to detect theCYP2C19*2and*3finally. On this basis of the technology, we detect the CYP19*2and*3alleles in104patients then compared with the sequencing results to certificate thereliability of this method.2In view of lack of low-specifity in single-phosphorothioate primersamplification system, we investgate the impact that Tth-SSB used as PCR additive inthe amplification system. Tth-SSB plasmid is constructed and expresse the protein, thenthe expressed Tth-SSB is added into PCR system to determine optimal usage andcondition, then consider improve specificity effectiveness. At last, we test CYP2C19*2and*3by established methods and compare with double-phosphorothioate primers to verify the accuracy of Tth-SSB as an additive into PCR for SNP screening. Inexperiments, dimethyl sulfoxide. betaine and trehelose as PCR additives are also addedinto the same amplification system respectively, compare the result with Tth-SSBamplification effectiveness.Results:1We have established a high-sensitivity and simply experiment technology thatcould be used for CYP2C19*2and CYP2C19*3alleles genotyping. The CYP2C19*2(681G> A) and CYP2C19*3(636G> A) genotyping results in104samples consistentwith parallel sequencing results completely. Show that this method is simple andreliable which can be used for clinical guidance of clopidogrel.2.Acquiring initially CYP2C19*2alleles of the three genotypes which A/A,G/Aand G/G frequency are8.65%,36.54%and54.81%, CYP2C19*3alleles of the threegenotypes which A/A,G/A and G/G genotype frequency are0.96%,9.62%and89.42%in Suzhou region.3The1μg/25μL Tth-SSB could improve the specificity and reduce the dimerwhen Tth-SSB is added into the PCR system.104patients genotyping resultscoincidence100%with double-phosphorothioate primers amplification system.4There are no effectivenesses in the amplification which dimethyl sulfoxide.betaine and trehelose are also added into the same amplification system respectively.Conclusions: Bases on "molecular switch" technology which3′double-phosphorthioate specific primers combination with high-fidelity Pfu DNApolymerase and add1μg/25μL Tth-SSB into single-phosphorothioate primersamplification system also could obtain accurate screening results in CYP2C19*2(681G>A) and CYP2C19*3(636G> A) alleles. The method simple and reliable andcould be used in clinical guidance of Clopidogrel.