实验二PCR乙肝RAPD.ppt

1、实验二PCR扩增乙肝病毒基因RAPD技术及基因多态性分析,PCR 可以把 指定的基因片段 数量放大,染色体,聚合酶链反应(polymerase chain reaction, PCR),引物,延伸,5,5,3,3,变性、退火,Taq polymerase Templet DNA dNTP primer Buffer,PCR reaction,耐热DNA聚合酶 模板DNA dNTPs 特异性引物 缓冲液,变性 95C,延伸 72C,退火 Tm-5C,PCR仪,PCR原理及相关知识,PCR实验操作,乙肝检测PCR反应,实验结果,琼脂糖凝胶板制备,两个实验的PCR产物,电泳检测,上午,下午,PCR实

2、验操作,RAPD PCR反应,血清样品处理,问 题:,1 PCR的技术特点? 2 PCR的应用价值? 3 基因多态性的意义 ?,实验分组:,一份用自制DNA模版做RAPD,2人1组,一份HBV-PCR反应,HBV反应管,RAPD反应管,(一)HBV试剂盒PCR检测血清样本 实验操作及注意事项,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HBV,1、待测血清中DNA简单制备,待测血清40ul + 40ul DNA 提取液， 混匀 沸水浴10m

3、in，10000rpm5min 取上清2l,样品模板管（可用做检测）,40ul DNA 提取液 沸水浴10min，10000rpm5min 取上清2l,阴性反应管 （做阴性对照）,阳性模板液 取2l,阳性反应管 （做阳性对照）,阳性反应1个,阴性反应1个,样品检测反应N个,阳性模板2ul,空白模板2ul,6000rpm5s，混匀，上PCR仪,每 行 座 位:,第一组,第二组,其余组,933min 预变性 94，50s 55，50s 72，50s 72，10min,30个循环,PCR反应程序：,（二）RAPD技术及基因多态性分析 实验操作及注意事项,RAPD实验PCR操作 每个RAPD反应：,6

4、000rpm5s，混匀，上PCR仪,932min 预变性 94，50sec 36， 50sec 72， 1min 72，5min,40个循环,RAPD的PCR反应程序：,琼脂糖凝胶板的制备 （封板、1%胶、凝固后拔梳）,点样,电泳（80100mA 25min） 紫外下观察结果,DNA琼脂糖凝胶电泳,RAPD,HBV,RAPD,HBV,四块胶点样分配：,点样顺序（ RAPD检测胶）,1 2 3 4 5 6 7 8 9 10 11 12,点样顺序（HBV检测胶）,第一块,1 2 3 4 5 6 7 8 9,第二块,1 2 3 4 5 6 7 8 9,对照,对照,对照,对照,PCR反应结果,PCR基

5、本原理及相关知识,The Polymerase Chain Reaction,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 finger

6、printing to mapping the human genome. 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

7、 can generate 100 billion similar molecules 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.,(1944 - ),The Nobel Prize in Chemistry 1993 for his invention of the polymerase chain reaction (PCR) method（1/2 of the prize

8、 ）,3 step and 30 cycles for PCR,Step 1. Denaturation,Step 2. Anneal,Step 3. Extend,30 cycles,create over a billion copies of the sequence,Step 1. Denaturation,The reaction is first heated to denature (separate) the sides of the double- stranded DNA,Temperature: 95 degrees Celsius,Step 2. Anneal,then

9、 cooled to allow the primers to find and bind to their complementary sequences on the separated strands.,Temperature: 55 degrees Celsius,Step 3. Extend,the polymerase to extend the primers into new complementary strands.,Temperature: 75 degrees Celsius,Step 4: Repeat the Cycles,Repeated heating and

10、cooling cycles multiply the target DNA exponentially, since each new double strand separates to become two templates for further synthesis.,This process can then be repeated up to 30 times to create over a billion copies of the sequence.,Amplification graph,What is in the PCR Mix?,Template (dsDNA) P

11、rimers (TWO short DNA: 15-30 nucleotides in length. Longer primers provide higher specificity ) P22 Taq polymerase dNTP Mix (dATP, dGTP, dTTP, dCTP) Reaction Buffer (including Mg2+),Taq polymerase,First, because the heat（ 95 degrees Celsius for denaturing） also caused the polymerase to split, the po

12、lymerase had to be replenished after every step, making the process expensive and significantly slower. Afterward the solution was to use Taq polymerase, derived from Thermus aquaticus, a bacterium that is native to the hot springs of Yellowstone. This was able to withstand the heat (up to 95 degree

13、s Celsius) without denaturing. This higher temperature allows a more uniform result to be produced. These improvements helped to made PCR cost effective., 影响PCR的主要因素 PCR反应体系的各个组分 PCR循环的温度（预变性、变性、退火、延伸） PCR循环的次数和平台效应 操作环境,相对DNA克隆而言，这种方法的特点：操作简便、时间短、特异性高、灵敏度高、实用性强； 广泛的应用于医学诊断、分子生物学、分子克隆、基因诊断、法医学、考古学等各

14、个领域。,RAPD反应检测DNA多态性的原理,RAPD (Random amplified polymorphic DNA）,RAPD技术具有快速、方便、灵敏度高、检测容易的优点。 运用随机引物扩增寻找多态性DNA片段可作为分子标记。用于检测DNA多态性。 建立于PCR技术基础上，利用一系列(通常数百个)不同的随机排列碱基顺序的寡聚核苷酸单链(通常为10聚体)为引物，对所研究基因组DNA进行PCR扩增。检测扩增产物DNA片段的多态性。 扩增产物DNA片段的多态性反映了基因组相应区域的DNA多态性。,RAPD所用的一系列引物DNA序列各不相同，但对于任一特异的引物，它同基因组DNA序列有其特异的结合位点。这些特异的结合位点在基因组某些区域内的分布如符合PCR扩增反应的条件，即引物在模板的两条链上有互补位置，且引物3 端相距在一定的长度范围之内，就可扩增出D