聚合酶链式反应

1、Polymerase Chain Reaction (PCR)聚合酶链式反应 After the experiment is finished, students should be able to explain the principle of Polymerase Chain Reaction (PCR) and know how to run a PCR experiment. 一、实验目的(Experimental Purpose ) 通过本实验学习PCR反应的基本原理与实验技术。二、实验原理(Experimental Principle) PCR was invented by K

2、ary Mullis and his colleagues in the 1980s, and is a relatively simple technique by which a DNA or cDNA template is amplified many thousand or million fold quickly and reliably. PCR技术是Kary Mullis和他的同事于20世纪80年代发明的。该技术相对较为简单，它可以快速可靠地将DNA或cDNA模板扩增数千倍甚至上百万倍。The polymerase chain reaction (PCR) is used to

3、 amplify a sequence of DNA using a pair of oligonucleotide primers each complementary to one end of the DNA target sequence. These are extended towards each other by athermostable DNA polymerase in a reaction cycle of three steps: denaturation, primer annealing and polymerization. 聚合酶链式反应(PCR)用于利用一对

4、寡核苷酸引物扩增一段DNA序列，其中每条引物分别与靶序列的两端互补。这对引物通过三步循环反应在热稳定的Taq DNA 聚合酶的作用下各朝着对应的方向延伸，每步循环反应包括以下三个步骤：模板变性，引物退火和引物延伸。 As figure explains, this technique uses enzyme (DNA polymerase) to make a copy of a defined region of DNA . First a high temperature (about 95) is needed to separate the DNA strands; 首先，高温(约95

5、 )使 DNA双链解离。Second a relatively low temperature (about 55) is needed to allow the primers to anneal to the template DNA strands; 其次，在相对较低的温度(约55)下使引物与模板DNA链退火。Third a medium temper-ature (about 72) is needed to allow DNA synthesis. 然后，在适中的温度(约72 )下使DNA合成。 Each cycle takes as little as a few minutes,

6、 and it usually rakes fewer than 30 cycles to produce as much amplified DNA as necessary.The PCR cycle: (1) The reaction cycle comprises a 95C step to denature the duplex DNA (2) an annealing step of around 55C to allow the primers to bind (3) a 72C polymerization step. Mg2+ and dNTPs are required i

7、n addition to template, primers, buffer and enzyme.A typical amplification reaction includes Primers (引物) Template DNA (模板DNA ) Taq DNA Polymerase (Taq DNA聚合酶) Nucleotides （寡核苷酸, dNTP） PCR buffer (PCR缓冲液)三、试剂与器材（Reagents and apparatus）. Instruments1. PCR Amplifier （ PCR 仪） 2. Electrophoresis System（

8、电泳系统）3. Ultraviolet transilluminator（紫外透射仪）4. Clean Benches （超净工作台） 5. Autoclave （高压灭菌锅） 6. Pipettes （微量加样器）. Reagents1. Sterile water 无菌水2. 10PCR buffer 10PCR缓冲液3. 25 mM MgCl2 氯化镁 4. 10 mM dNTPs 单核苷酸5. 50M oligonucleotide primers 1 677F 引物16. 50M oligonucleotide primers 2 677R 引物27. 5 unit/l Taq DN

9、A polymerase Taq DNA 聚合酶8. Template DNA 模板DNA四、实验步骤(Experimental Procedures)1.Combine the following for each reaction in a PCR tube (0.2ml） 在PCR (0.2ml）小管中将下列各组分混合：2. Prepare a control reaction with no template DNA and an addition 35.5l of sterile water. 设立对照反应，其中不含模板DNA，而加35.5 l 无菌水。3. Run the following program: 按以下程序运行95 preheated 8 min 95 1 min.62 1 min.72 1 min for 36 cycles.Program a final extension at 72 for 7 min.PCR AmplifierStart