| Objective: Cytochrome P450 super family are important enzymes metabolizing a serials of endogenic and ectogenic chemistry, including many drugs, about 60% of drug in use are cleared by the involving of CYP450s. The activity of Cytochrome P450 is significantly different in various races and groups. The polymorphism and the genetic variance intimately induced the drug adverse reaction (ADR). The polymorphism of CYP1A2, CYP2D6, CYP2C19, CYP2C9, CYP3A4, and CYP3A5 in CYP450 closely relates with the great majority of ADR. The mutant of CYP3A4 rarely occurred in Asian, less than 1%. Furthermore, the mutant of CYP3A4 was not founded during the bolting of the standard preparation. Therefore we chose CYP1A2*1F, CYP1A2*1C, CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP2D6*10, CYP2D6*2, CYP3A5*3 as targets which mutant occurred frequently in Asian. In this paper, the rapid, accurate, high-throughout DNA miroarray technology was developed for detection and identification of 8 SNPs preceding. These studies provided effective diagnosis tools for clinical diagnosis, medication guide to prevent the ADRs.Methods: Eight SNPs mentioned from CYP450 were chosen as targets for screening of specific probes and designing primers to detect and identify five CYP450 hypotypes. The duplex PCR reaction condition was optimized. Furthermore , in these studies, the factors were investigated which effect on hybridization, such as anticoagulant, extraction DNA form blood, Mg2+ concentration, components of hybridization buffer etc. Wild-type plasmids and mutant-type plasmids were constructed as quality control standard substances. On the basis of screening standard substances from five hundred samples and the duplex PCR reaction, the Cutoff values according to DNA microarray assay were confirmed. Furthermore, specificity, sensitivity and reproducibility of microarray assays were determined respectively.Results: The target alleles were CYP1A2*1F, CYP1A2*1C, CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP2D6*10, CYP2D6*2, CYP3A5*3. Sequences of specific primers and probes were determined. EDTA or natrium citricum was chosen as suitable anticoagulation. The suitable method for extracting genomic DNA was modified salt fractionation. Wild-type plasmids and mutant-type plasmids were constructed successfully. The optimal conditions of PCR reaction were as follows: 1) CYP1A2*1F combine with CYP3A5*3. The concentration ratio of forward primer to reverse primers labeled with Cy3 was 1:10, and the final concentration of CYP1A2*1F-forward primer and CYP3A5*3-forward primer was 1.25μmol/L and 0.75μmol/L accordingly. The concentration of Mg2+, Taq enzyme, and dNTPs was 1.5 mmol/L, 1.5U, 100μmol/L respectively. 2) CYP2C9*3 combine with CYP2D6*2. The concentration ratio of forward primer to reverse primers labeled with Cy3 was 1:10, and the final concentration of CYP2C9*3-forward primer and CYP2D6*2-forward primer was 0.75μmol/L and 1μmol/L accordingly. The concentration of Mg2+, Taq enzyme, and dNTPs was 1.5 mmol/L, 2.0U, 100μmol/L respectively. 3) CYP2C19*3 combine with CYP2D6*10. The concentration ratio of forward primer to reverse primers labeled with Cy3 was 1:10, and the final concentration of CYP2C19*3-forward primer and CYP2D6*10-forward primer was 1.25μmol/L and 1μmol/L accordingly. The concentration of Mg2+, Taq enzyme, dNTPs was 2.0 mmol/L, 1.0U, and 100μmol/L respectively. 4) CYP1A2*1C combine with CYP2C19*2. The concentration ratio of forward primer to reverse primers labeled with Cy3 was 1:10, and the final concentration of CYP2C1A2*1C-forward primer and CYP2C19*2-forward primer was 1.0μmol/L and 0.75μmol/L accordingly. The concentration of Mg2+, Taq enzyme, dNTPs was 3.0 mmol/L, 2.0U, and 100μmol/L respectively. The optimal annealing temperature and time was 56℃and 30s respectively. The suitable conditions of hybridization reaction were as followed: hybridization temperature was 42℃, and components of hybridization buffer were 8×SSC, 0.2%SDS, 2.5% formamide. Wild-type and mutant-type standard substances were identified through duplex PCR from five hundred samples of blood. On the basis of these researches, the Cutoff values according to DNA miroarray assays were determined respectively. Accuracy rating of DNA microarray was 100%, when sequencing the standard substances randomly. The limit of detection was approximately 1ng/μl of DNA. The reproducibility of the assay was also investigated, and it showed the coefficient of variation of intra-assay and inter-assay was less than 15%.Conclusions: DNA microarray for detection of cytochrome P450 hot mutant in Asian is high-throughout, rapid, sensitive, specific assay having a very good reproducibility and specificity. This chip was applied to clinical diagnosis and guidance medicine as a preliminary screening tool. It will be valuable for enhancing the diagnosis level and reducing the drug adverse reaction.
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