研究生课程：PCR技术的新进展及其应用.pptVIP

Kary Mullis 1983,POLYMERASE CHAIN REACTION,A licence to do molecular biology A key central technique that has revolutionised molecular and consequently cell biology,In this Lecture,Principle of PCR PCR, the basics Optimize the PCR and troubleshoot Application of PCR Reverse-Transcription PCR（反转录PCR PCR cloning（PCR克隆技术 Real-Time PCR（实时定量PCR History of PCR,Polymerase Chain Reaction,PCR是一种体外酶促合成特定DNA片断的技术，是根据人类的需要对复杂生命过程的一种简单化的模拟。PCR技术的原理是DNA半保留复制。,DNA Replication in vivo,/AB/GG/collaboration.html,Strand separation Synthesis of short RNA primers Synthesis of new DNA strands,DENATURATION 93C - 95C,DNA replication in vitro,The PCR Process in vitro,Template DNA: The sample to amplified Primers Short, specific segements of DNA, provide SPECIFICITY dATP, dTTP, dCTP, dGTP Thermostable DNA polymerase (e.g., Taq, Pfu) Buffer and salts (KCl, MgCl2) Optional: BSA, DMSO, Formamide,TYPICAL REACTION MIXTURE,25 or 50ls in a micro Eppendorf (0.5ml) tube,(1.0-4.5 mM),The PCR in vitro,/resources/BiologyAnimationLibrary.htm,PCR,Agarose gel electrophoresis,The final product,UV visualisation,3-4 hours,Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva, semen Thermo-stable DNA polymerase e.g. Taq polymerase Oligonucleotides Design them well! Buffer Tris-HCl (pH 7.6-8.0) Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP),OPTIMISING PCR THE REACTION COMPONENTS,Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva, semen Thermo-stable DNA polymerase e.g. Taq polymerase Oligonucleotides Design them well! Buffer Tris-HCl (pH 7.6-8.0) Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP),OPTIMISING PCR THE REACTION COMPONENTS,Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva, semen Thermo-stable DNA polymerase e.g. Taq polymerase Oligonucleotides Design them well! Buffer Tris-HCl (pH 7.6-8.0) Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP),OPTIMISING PCR THE REACTION COMPONENTS,Thermastat DNA polymerase: Taq, Pfu, Vent e.g.,Isolated from a thermophilic bacterium (Thermus aquaticus) from a culture derived from a hot spring in Yellowstone. Catalyzes DNA polymerization at elevated temperature (72C) and is resistant to 98 C. Similar enzymes have have been identified from other thermophilic bacteria, such as those living in deep sea vents.,Number of options available Taq polymerase Pfu polymerase Tth polymerase How big is the product? 100bp 40-50kb What is end purpose of PCR? Sequencing/mutation detection-Need high fidelity polymerase Cloning (TA cloning)-Taq DNA Polymerase,CHOOSE YOUR POLYMERASE WITH CARE,Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva, semen Thermo-stable DNA polymerase e.g. Taq polymerase Oligonucleotides Design them well! Buffer Tris-HCl (pH 7.6-8.0) Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP),OPTIMISING PCR THE REACTION COMPONENTS,Length 18-30nt (21nt) Base composition; 50 - 60% GC rich pairs should have equivalent Tms Tm = (number of A+T residues) x 2 C + (number of G+C residues) x 4 C Initial use Tm5C Avoid internal hairpin structures no secondary structure Avoid a T at the 3 end Avoid overlapping 3 ends will form primer dimers Can modify 5 ends to add restriction sites etc,PRIMER DESIGN IS VITAL,optimise for each set of primers,OPTIMISE PCR CONDITIONS-Primer,X,DNA primers for PCR,PRIMER DESIGN,Primer5 from Premierprimer Co.,Also available on internet http:/www.hgmp.mrc.ac.uk/GenomeWeb/nuc-primer.html OligoSys: ,Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva, semen Thermo-stable DNA polymerase e.g.Taq polymerase Oligonucleotides Design them well! Buffer Tris-HCl (pH 7.6-8.0) Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP),OPTIMISING PCR THE REACTION COMPONENTS,TITRATE YOUR Mg2+ CONCENTRATION!,4 3.5 3 2.5 2 1.5 1 mM,Normally, 1.5mM MgCl2 is optimal Best supplied as separate tube Always vortex thawed MgCl2 Mg2+ concentration will be affected by the amount of DNA, primers and nucleotides,USE MASTERMIXES WHERE POSSIBLE,Taken from -/genetics/ward/tavi/PCR.html,PCR Optimization,Focus on temperature and MgCl2,THE PERFECT RESULT,APPLICATIONS OF PCR,基因克隆： 基因表达的定量：半定量RT-PCR和实时定量RT-PCT 反转录PCR，RT-PCR 快速扩增cDNA 末端 RACE DNA序列分析,已知序列基因的PCR研究,代表性差异显示PCR: RDA 差异显示RT-PCR:：DDRT 快速扩增多态性DNA：RAPD,未知序列基因的PCR研究,PCR技术 在基因克隆 基因表达差异分析中常用的方法,PCR cloning Semi-quantitative RT-PCR Real-Time PCR,Cloning of PCR products,Modified primers by adding RE sites at the 5end of each primer T-A strategy: because some thermostatic DNA polymerase can add a non-template-depended A at the 3end of PCR products, so can inserted the PCR products into a T-vector diractly Blunt-end PCR products cloning, many thermostatic DNA polymerase can amplify the DNA correctly, Inserted the DNA products into blunt-RE digested plasmid diractly.,DNA聚合酶的特性及在基因克隆中的应用,Taq DNA聚合酶：某些耐热的DNA聚合酶，在DNA链延伸到模板DNA的末端时，并不是立即终止新链的延伸，而是在新链的3末端加上一个A，形成突出的3末端，为基因克隆提供了一个天然的粘末端。,逆转录酶：来自于逆转录病毒的逆转录酶，在以mRNA为模板，反转录cDNA时，当新链延伸到模板的末端时，并不是立即终止新链的延伸，而是在新链的3末端加上几个CCCC，形成突出的3末端，为合成全长的cDNA提供了一个天然接头位点。,PCR T-A cloning,PCR,T T,PCR cloning T-A strategy,NNNGGATCC,TCTAGAMMM,NNNGGATCC YYYCCTAGG,AGATCTMMM TCTAGAWWW,EcoRI和BglII,GATCC G,A TCTAG,相应酶切的克隆质粒,PCR 克隆：通过引物对克隆片断添加酶切位点,T4 DNA酶 连接,NNNGGATCC,TCTAGAMMM,NNNGGATCC YYYCCTAGG,AGATCTMMM TCTAGAWWW,EcoRI和BglII,GATCC G,A TCTAG,相应酶切的克隆质粒,PCR 克隆：通过引物对克隆片断添加酶切位点,T4 DNA酶 连接,RT-PCR,RTase: Avian myeloblastosis virus(AMV) and Moloney strain of murine leukemia virus(MMLV). Primers for first-stranded cDNA synthesis: Gene specific primers, Oligo(dT) primers for binding the poly(A) mRNA and Random hexamer primers,RT-PCR,To amplify cDNA copies of RNA To retrieve and clone the 5 and 3 terminus of RNA To generate cDNA library from a very small amounts of mRNA To identify the mutations and polymorphisms To measure the strength of gene expression (semi-quantitative RT-PCR),RT-PCR,Full length cDNA synthesis with PCR,(Switching Mechanism At the 5 end of the RNA Transcript) technology,RT-PCR,RTase: Avian myeloblastosis virus(AMV) and Moloney strain of murine leukemia virus(MMLV). Primers for first-stranded cDNA synthesis: Gene specific primers, Oligo(dT) primers for binding the poly(A) mRNA and Random hexamer primers,基因表达差异的分析技术,Northern Blot Western blot Semi- quantatitive RT-PCR DNA Chip Protein Chip 二维电泳 质谱,NORTHERN,target gene,internal control gene actin, GAPDH, RPLP0 etc,10X,2X,control,expt,Corrected fold increase = 10/2 = 5,Semi-quantitative RT-PCR,Corrected fold increase = 810 问题:难以准确设定PCR扩增的平台期,Real-Time PCR Instruments,dilutions target DNA,dilutions reference DNA,C,C,C,C,C,C,E,E,E,E,E,E,target primers,reference primers,triplicates cDNA,triplicates cDNA,Standard curve method,Differences between basic and Real-Time PCRs,Semi-quantitative, not sensitivity PCR products vary from 100 to several kbps,Real-time detection: Each cycle produces a fluorescent signal Absolute quantitative, much more sensitivity PCR products are around 60-150 bps,Semiquantatative PCR,Real-Time PCR,Real-Time PCR -Taqman probe,ABI PRISM 7000 and 7700 Sequence Detection System Real-time detection: Each cycle produces a fluorescent signal proportional to the amount of PCR product present,“Real-Time” PCR,F,Q,.,.,2. Laser excites fluorescent tag on primer.,3. Fluorescence is absorbed by quencher molecule,4. Detector registers “no reaction”.,“Real-Time” PCR,F,Q,.,.,T,.,5. Primer is extended by Taq Polymerase,“Real-Time” PCR,F,Q,.,.,T,.,.,6. Extension continues until the Polymerase Meets the fluorescent molecule.,7. The polymerase breaks down the tagged primer. The fluorescent tag and quencher molecule become separated.,“Real-Time” PCR,F,Q,.,T,.,.,.,Q,Q,F,F,8. As PCR continues, the tagged primers are degraded and “free” fluorescent molecules accumulate.,9. The detector records fluorescence as a measure of amplification progress.,Real-Time PCR-SYBR green,SERIES OF 10-FOLD DILUTIONS,IL1-b con,IL1-b vit,av =18.03,av =29.63,IL1-beta,Real-Time PCR,Taqman probe Specific primers Fluorescent dye labeled probe Expensive High specificity,SRBR green Specific primers No probes Cheaper Reliable,History of PCR,PCR技术是由美国科学家Kary Mullis于1983年发明的。由于当时没有耐热DNA聚合酶，因此PCR过程中每个循环都需要添加DNA聚合酶，PCR技术操作十分烦琐而且不实用。 PCR技术的自动化归因另外2个重大发现：耐热DNA聚合酶的发现，和计算机控制的热循环仪（DNA thermal cycler)的出现。 Taq DNA聚合酶被美国Science杂志评为1993年的明星分子。,History of PCR,1987年PCR技术得到美国专利局的专利授权。 1989年DuPont公司对该专利提出异议：PCR技术应当是公共产权。 斯坦福大学的Kornberg HG也对PCR技术专利提出异议。理由是认可具有生物学知识的人都可以从Kornberg的文章推知如何操作PCR。 1993年Mullis Kary获得诺贝尔奖。,History of PCR,Milestones of PCR,DNA polymerase: Kornberg A.(1957) Oligonucleotide synthesis: Khorana HG (1967-1968) The PCR idea: Khorana HG(1971, J. Mol.Biol) The idea: Kary Mullis(1983) Thermostable DNA polymerase(1989) Automated Thermal cycler Innovative applications Real-Time PCR RACE Flow Chip PCR,History of PCR,Louis Pasteur: Once remarked that “chance favors the prepared mind,“ and certainly the history of scientific progress supports his contention, such as Newtons discovery of gravity following his encounter with an apple, Flemings discovery of penicillin on a contaminated petri dish. Kary Mullis: Scientists today continue to take unexpected turns on their paths to discovery. One such recent detour occurred in 1983 on U.S. Route 101 in northern California.,History of PCR,Kary Mullis was born in 1944 in North Carolina. He obtained his Bachelors degree in Chemistry in 1966 from the Georgia Institute of Technology and in 1972 received a PhD in Biochemistry from the University of California at Berkeley. He was then offered a technicians post at the Cetus Corporation of Emeryville in 1978. In 1983, whilst driving along the highway from San Francisco to his home in La Jolla, California, Mullis was thinking about a simple method of exponentially amplifying a DNA sequence in a test tube. Mullis then took his concept to his associates at Cetus and together they took the idea and made it work in an experimental system, Mullis was awarded 10,000 US