头发中抽提DNA课件.ppt

头发中抽提DNA并用PCR- RFLP法 鉴定CYP2C9基因型 1.感受科学研究 2.接触科学研究 3.自己动手做科学研究（由简单到复杂） 本科生---硕士生---博士生 学习---研究---创新 新发现、新方法、新结论 Homework mgkosff 2005/5/8 Index Part 1 review of two papers Part 2 discussion of my own (emphasis) Part 3 some information Part 4 supplement Part 1 Review of two papers Identification of a novel variant CYP2C9allele in Chinese Part 1 Review of two papers Background information: Cytochrome P450 2C9 (CYP2C9), a member of the CYP2C enzyme subfamily, ranks among the most important drug metabolizing enzymes in humans. Genetic polymorphism (SNP) of CYP2C9: wild-type protein CYP2C9*1 CYP2C9*2 (Arg144Cys), CYP2C9*3 (Ile359- Leu), CYP2C9*4 (Ile359Thr), CYP2C9*5 (Asp360Glu), CYP2C9*6 (null allele) and 5’-noncoding region Tolbutamide is widely accepted as a probe substrate for the assessment of hepatic CYP2C9 activity Part 1 Review of two papers Methods and results: Tolbutamide pharmacokinetics(The procedures is omitted) The PM study subject’s plasma half-life is about 9–15 times that of the CYP2C9*1/*1 or CYP2C9*1/*3 carriers and is even slower than the CYP2C9*3/*3 genotype subjects. Sequencing of CYP2C9(CYP2C9 gene was amplified by PCR) and Genotyping tests (PCR–RFLP analysis) Revealing the study subject is heterozygous for CYP2C9*3 and a previously unreported mutation in exon 2. This mutation associates with a T269C transversion of the CYP2C9 gene that leads to a Leu90Pro substitution in the encoded protein Part 1 Review of two papers Frequency analysis (PCR–RFLP analysis---The T269C change in the sequence CTTGG creates a new recognition site of EcoRII (CCTGG) such that EcoRII digested the PCR products of the CYP2C9 T269C allele into fragments of 192 bp, 195bp and 302 bp, whereas the PCR products of the CYP2C9*1 allele were digested into fragments of 387 bp and 302 bp) These data correspond to an allele frequency of 1.02% (95% confidence limits of 0.2 to 3.0%) in the Chinese population. Part 1 Review of