PCR-I-XXXX10-bin.pptx

Principle&Practice of PCR 罗罗 彬彬 Dept.Histolgy&Embryology Welcome!成绩评定成绩评定 实验课考勤实验课考勤 笔试笔试(开卷）开卷）课程安排课程安排 理论课（理论课（6W6W-8W8W）：）：W2/3W2/3晚上晚上 实验课（实验课（9W9W-11W11W）：）：W1/2/4/5W1/2/4/5全天全天 W3W3下午下午 办公地点：办公地点：104104馆馆3 3楼楼 科室电话：科室电话：53585775358577 EmailEmail： The proof of the pudding is in the eating.Knowledge is a treasure,but practice is key to it.Introduction about PCR Part I -What is PCR?-How did the PCR develop?-What do we need for doing the PCR?-How did the PCR work?-What is the PCR used for?Outline -What is PCR?-How did the PCR develop?-What do we need for doing the PCR?-How did the PCR work?-What is the PCR used for?Outline What is PCR?Polymerase Chain Reaction (聚合酶链反应聚合酶链反应)A method to amplify selected sections of DNA in vitro(选择性体外扩增选择性体外扩增DNA片段的方片段的方法法).A process which“Amplifies”or“Copies”a piece of DNA repeatedly until there is an amount which is great enough to observe visually.DNA replication vs.PCR PCR is a laboratory version of DNA replicaiton and commonly called“in vitro”since it occurs in a test tube while“in vivo”signifies occurring in a living cell.-What is PCR?-How did the PCR develop?-What do we need for doing the PCR?-How did the PCR work?-What is the PCR used for?Outline *1930*1930年正式提出年正式提出核酸核酸的概念的概念 *1953*1953年年Watson&Crick Watson&Crick 提出提出DNADNA双螺旋双螺旋 结构结构及及半保留复制半保留复制模型模型 Har Gobind Khorana *19711971年年KhoranaKhorana最早提出最早提出核酸体外扩增核酸体外扩增的设想的设想 经过经过DNADNA变性，与合适变性，与合适的引物杂交，用的引物杂交，用DNADNA聚合酶聚合酶延伸引物，并不断重复该过延伸引物，并不断重复该过程便可克隆程便可克隆tRNAtRNA基因基因 1968年诺贝尔生理学或医学奖年诺贝尔生理学或医学奖 “I do my best thinking while drivingI do my best thinking while driving”The idea was not the product of a painstaking laboratory discipline,but was conceived while cruising on highway.For this brilliant idea,a$10,000 bonus from Cetus.*1985*1985年年 Kary Mullis Kary Mullis 发明了发明了PCRPCR received Nobel Prize for chemistry in 1993.PCR has become the most widely used nucleic acid amplification technology and integral part of molecular biology.-from the scientific method to parapsychology,-from poisonous spiders to the HIV virus and AIDS,-from global warming to astrology,-from the O.J.Simpson trial to how you can turn a light bulb on with your mind.Dancing Naked in the Mind Field In the book he writes with passion and humor about a wide range of subjects:-What is PCR?-How did the PCR develop?-What do we need for doing the PCR?-How did the PCR work?-What is the PCR used for?Outline 5 Essential Components of PCR Reaction salts(ionssalts(ions)dNTPsdNTPs PrimersPrimers DNA DNA PolymerasePolymerase TemplateTemplate What do we need for doing the PCR?Basic Components:*Template(模板模板)：DNA,cDNA;*Primer(引物引物):oligonucleotides(寡核苷酸寡核苷酸);*dNTP(三磷酸脱氧核苷酸三磷酸脱氧核苷酸)*DNA Polymerase(聚合酶)*Buffer(缓冲液缓冲液)Are you a good cook?delicious Deoxyribonucleic Acid(DNA)Complementary DNA(cDNA):single-stranded DNA made from a messenger RNA template with reverse transcriptase (逆转录酶）逆转录酶）.1.Template(模板模板)：Previously isolated and purified Short preexisting oligonucleotides to which new deoxyribonucleotides can be added by DNA polymerase Two primers have to flag the beginning and end of the gene to be copied and are complementary to it 待扩增待扩增 DNADNA片段两翼互补的寡核苷酸片段两翼互补的寡核苷酸 Primers provide specificity 5 3 3 5 Primer 1 Primer 2 2.Primer(引物引物):oligonucleotides(寡核苷酸寡核苷酸);The Size of the DNA Fragment Produced in PCR is Dependent on the Primers The PCR reaction will amplify the DNA section between the two primers.If the DNA sequence is known,primers can be developed to amplify any piece of an organisms DNA.Forward primer Reverse primer Size of fragment that is amplified 3.Deoxynucleotide triphosphates（dNTP）:Provide energy and nucleosides for the synthesis of DNA Adenine（腺嘌呤腺嘌呤）Thymidine（胸腺嘧啶胸腺嘧啶）Cytosine（胞嘧啶胞嘧啶）Guanine（鸟嘌呤鸟嘌呤）4.DNA Polymerase(聚合酶)Heat-stable polymerase is vital to the ease of the process Taq Polymerase An enzyme from Thermus aquaticus that survives in hot springs in Yellowstone National Park and is capable of growing in 70-75；4.DNA Polymerase(聚合酶)Original report was published in 1976;Roughly 10 years later,PCR was developed.a thermostable enzyme;catalyzing the polymerization of nucleotides into duplex DNA in the 5-3 direction;53 DNA polymerase;lacking 3 5 exonuclease activity;having error rate.DNA polymerase needs Mg+as cofactor;Each DNA polymerase works best under optimal temperature,pH and salt concentration;PCR buffer provides optimal pH and salt condition.5.Buffer -What is PCR?-How did the PCR develop?-What do we need for doing the PCR?-How did the PCR work?-What is the PCR used for?Outline How did the PCR work?3 conceptsconcepts 3 steps3 steps One sentenceOne sentence 3 Concepts of the PCR 1.Denaturation:变性变性 2.Renaturation：复性复性 3.Semiconservative replication：半保留复半保留复制制 the double strand DNA open to single stranded DNA two single stranded DNA form double strand DNA replication,one make two SemiSemi-conservative replicationconservative replication Old new 3 steps d-a-e cycle 变性变性 退火退火:time?/?延伸延伸 opening double-strand DNA at 94 C Making single-strand DNA accessib