聚合酶链式反应

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,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 Kary 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 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 ),使,DNA,双链解离。,S,econd,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, 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.,Mg,2+,and dNTPs are required in 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,）,.,Instruments,1. PCR Amplifier,（,PCR,仪）,2.,Electrophoresis System,（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）,. Reagents,1. Sterile water,无菌水,2. 10PCR buffer,10PCR,缓冲液,3. 25 mM MgCl,2,氯化镁,4. 10 mM dNTPs,单核苷酸,5. 50,M oligonucleotide primers 1,677F,引物,1,6. 50,M oligonucleotide primers 2,677R,引物,2,7. 5 unit/,l,Taq,DNA 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 35.5,l,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 Amplifier,Start,Files,Options,Lid,Incubate,样品,DNA,：,16ul,上样缓冲液：,4ul,Mark DNA,样品：,6ul,共,20ul,4. Electrophorese gel.,在,3,琼脂糖胶中进行电泳。,DNA Marker,PCR product,五、实验结果,(Experimental Results),M,