常用中药的生物鉴定.pptxVIP

常用中药的生物鉴定;常用中药的生物鉴定方法; 聚合酶链式反应 (Polymerase Chain Reaction)，简称PCR，是一种分子生物学技术，用于放大特定的DNA片段。可看作生物体外的特殊DNA复制。; 少量DNA 样品快速扩增amplify技术。 (invented by Kary Mullis , won a Nobel Prize in Chemistry in 1993 );原 理; PCR 要 件 模板Template 引物 Primers: Small pieces of DNA. Made up of 15 to 30 bases.? Binds the DNA at a specific region. forward and reverse 正向和逆向 酶Enzyme: Taq DNA polymerase PCR核酸Nucleotides: Substrate, four deoxyribonucleotides (A,C,G,T) dNTP ;M;取出15 ul ;;;;Program Denature 95°C 5 min Denaturation变性：双链DNA模板在热作用下， 氢键断裂，形成单链DNA，一般加热至90－95℃, 30 sec Annealing褪火：系统温度降低，引物与DNA模板结合，形成局部双链，55－70℃，固称其为褪火。 most important factor in the PCR ,45sec Extension延???：在Taq酶的作用下，以dNTP为原料，从引物的5′端→3′端延伸，合成与模板互补的DNA链, 温度为70－75oC, 1.5 min Final extension 72 °C, 5 min;;;;;;;;;;;;;2x106;优点: need very small amount of sample;Factors affect to PCR Mg +2 concentration, Mg +2 ? specificity ? 0.1~5mmol/L Mg+2 预实验 Primer(引物) specificity Degenerate primer ---to find the target gene in the closely related species specificity ?, nonspecific products ?;Annealing Temperature Too high no PCR product Too low much nonspecific product gradient Temp PCR machine find the suitable Temp Taq polymerase quality Template(模板) DNA quality ; PCR产物鉴定 Agarose gel electrophoresis to check product ;第30页/共47页;After PCR amplification Agarose gel electrophoresis to check product 1.5% agarose in 17 ml TAE ( 4mm thick) 5 ul product add 1 ul 6X loading dye mix 1Kb marker 2.5 ul 100 V run 25-30 min EB stain 10 min(EB致癌物, 戴手套) , photograph; 1.5 1.0 0.5 0.2 ;PCR的应用 1.detection genetically modification organism (GMO )检测转基因生物，找出特异的基因或片段 find specific gene or fragment (promoter) 2.uncultured microorganism 3.pathogen identification 4.detection insertion sequence (cloning) ;提取模板; 乌梢蛇为《中国药典》(2010版)一部收载的一种常用中药，来源于游蛇科动物乌梢蛇[Zaocys dhumnades(Cantor)]的干燥体。有祛风、通络、止痉之功效。用于风湿顽痹、麻木拘挛、半身不遂、抽搐痉挛、瘰疠恶疮。 商品流通中多以