分子医学技能课件-分子医学技能-实验二.pptx

1、PCR实验结果实验结果图图 Sometimes called molecular photocopying,the polymerase chain reaction(PCR)is a fast and inexpensive technique used to amplify,or make many copies of,small segments of DNA.The technology empowers scientists to undergo a number of processes,from DNA fingerprinting to mapping the human ge

2、nome.In 1983,Kary Mullis invented the PCR.The process is an invaluable tool to todays molecular biologists and biotechnology corporations.Kary B.Mullis As he wrote later in Scientific American:Beginning with a single molecule of the genetic material DNA,the PCR can generate 100 billion similar molec

3、ules in an afternoon.The reaction is easy to execute.It requires no more than a test tube,a few simple reagents and a source of heat.The Nobel Prize in Chemistry 1993 for his invention of the polymerase chain reaction(PCR)method（1/2 of the prize）3 step and 30 cycles for PCRStep 1.Denaturation（变性）The

4、 reaction is first heated to denature(separate)the sides of the double-stranded DNA Temperature:95 degrees Celsius Step 2.Anneal（退火）then cooled to allow the primers to find and bind to their complementary sequences on the separated strands.Temperature:55 degrees Celsius Step 3.Extend（延伸）the polymera

5、se to extend the primers into new complementary strands.Temperature:75 degrees Celsius Repeat the Cycles Repeated heating and cooling cycles multiply the target DNA exponentially,since each new double strand separates to become two templates for further synthesis.1个循环后的结果个循环后的结果第第2个循环后的结果个循环后的结果模版模版

6、第第1次的产物次的产物第第2次的产物次的产物AABBCD第第3个循环后的结果个循环后的结果第第3次的产物次的产物第第4个循环后的结果个循环后的结果第第4次的产物次的产物1A，1B，2C，2D，2EAB2C2D2E1A，1B，3C，3D，6（22+2）EAB3C3D6E第第N个循环后的结果个循环后的结果第第N次的产物次的产物1A，1B，（N-1）C，（N-1）D，(2n-2+2)E(2n-2+2)EABN-1CN-1DComponents for PCR Reaction Template(dsDNA)Primers(TWO short DNA:15-30 nucleotides in leng

7、th.Longer primers provide higher specificity)P22 Taq polymerase（Thermus aquaticus）dNTP Mix(dATP,dGTP,dTTP,dCTP)Reaction Buffer(including Mg2+)一般PCR实验设计：样品反应 不同的退火温度梯度 不同的Mg2浓度 不同的模板浓度 阴性对照反应 阳性对照反应HBV检测试剂盒介绍 构成：DNA提取液 检测反应管（PCR反应体系，上样buffer，石蜡油）阳性模板 设计：反应管中有针对HBV的特异性引物，若出现与阳性对照相同的结果，则说明待测样品中含有HBV1、待测血清中DNA简单制备上清上清PCR反应程序：反应程序：实验目的及预期结果 目的：检测来自不同样本的基因多样性 检测技术：apoB基因的PCR 预期结果：琼脂糖电泳条带的差异apoB多态性分析的基本实验过程来源不同的待测样本实验主要步骤 apoB基因 PCR，1人1份 分装PCR反应液每份21ul（包括：Taq，引物一对，反应缓冲液，dNTP）加入模板4ul（口腔细胞基因组DNA）混匀，离心 上PCR仪30个循环个循环HBV-PCR反应结果预测反应结果预测