1、 DNA Polymerase The polymerase chain reaction(PCR)is to used to amplify a sequence of DNA in vitro,using a pair of primers each complementary to one end of the the DNA target sequence.理论理论技术技术Denaturation(变性变性):The target DNA(template)is separated into two stands by heating to 95Primer annealing(退火退
2、火):The temperature is reduced to around 55 to allow the primers to anneal.Polymerization(elongation,extension)(延伸延伸):The temperature is increased to 72 for optimal polymerization step which uses dNTPs and requires Mg+.The principle of PCR:Three different steps proceed in each PCR cycle.5353535355335
3、3535353535353535353535355335535353353535353DenaturationAnnealingExtension(DNA polymerase)Cycle 1Cycle 2Cycle 3InitialDNA8421Number of DNA molecules(2n):指数扩增指数扩增Many cycles(25-35 in common)are performed to complete one PCR reaction,which resulted in an of the target DNA in which both forward and reve
4、rse primers pair.DNA template(模模板板)that provides one or more target molecules can in principle be used as a template for PCR.Whatever the source of template DNA,PCR can only be applied if so that primers can be designed.Figure 8-3 Substrates required for DNA synthesisPCR Primers1.Anneal on opposite
5、strands of the target sequence:forward and reverse primers（正向引物和反向引物）（正向引物和反向引物）2.Have similar G+C contents(Tm)so that they anneal to their complementary sequences at similar temperatures (anneal temperature:Tm-5C)5-ATTCCGATCGCTAATCGATGGC-TCCTGTGCA TTTCGCCACTAGAG-33-TAAGGCTAGCGATTAGCTACCG-AGGACACGTA
6、AAGCGGTGATCTC-55-ATTCCGATCGCTAATCGATG-33-CACGTAAAGCGGTGATCTC-5forward primerreverse primer5-CTCTAGTGGCGAAATGCAC-353535353553353535353535353535353535355335535353353535353DenaturationAnnealingExtension(DNA polymerase)Cycle 1Cycle 2Cycle 3PCR Enzymes Denaturation(变性变性):The target DNA(template)is separa
7、ted into two stands by heating to 95 Primer annealing(退火退火):The temperature is reduced to around 55 to allow the primers to anneal.Polymerization(elongation,extension)(延伸延伸):The temperature is increased to 72 for optimal polymerization step which uses dNTPs and requires Mg2+.Taq polymerase：isolated
8、from the thermophilic bacterium Thermus aquaticus(嗜热菌）嗜热菌）,耐热耐热DNA聚合酶，聚合酶，耐高温耐高温 It has no 3 to 5 proofreading exonuclease activity High-accuracy DNA polymerase is available commercially(高保真酶）高保真酶）PCR仪仪Discovery of PCR technique 希望大家不仅仅知道希望大家不仅仅知道“是什么是什么”，而且也，而且也了解了解“为什么为什么”和和“结论是怎么得出来的结论是怎么得出来的”如果你
9、的研究能力一般，那么，改革本研究领域中大家习以为常的最基本的技术，你就有可能获诺贝尔奖 -Kary Mullis(卡里卡里.穆利斯穆利斯)The replication of DNA 哥伦布竖立鸡蛋的故事哥伦布竖立鸡蛋的故事 DNA分子的拷贝术，复印机PCR技术的发现充满传奇色彩技术的发现充满传奇色彩Kary Mullis(卡里卡里.穆利斯穆利斯)1972年获得加州大学伯克利分校生物化学博士学位年获得加州大学伯克利分校生物化学博士学位1979年进入塞特斯公司：与年进入塞特斯公司：与DNA合成相关的工作合成相关的工作1983年年4月：月：PCR的最初想法的最初想法高速公路的灵感高速公路的灵感23
10、0=10亿亿想法付诸行动想法付诸行动1983年年12月：第一个月：第一个PCR反应成功反应成功1984年塞特斯公司申请年塞特斯公司申请PCR技术专利技术专利 PCR技术引来的官司技术引来的官司 谁发明了谁发明了PCR？DNA聚合酶的发现者？聚合酶的发现者？纸上谈兵纸上谈兵 Kary Mullis(卡里卡里.穆利斯穆利斯)胜诉胜诉Kary Mullis won the 1993 Nobel Prize in Chemistry for his invention of the polymerase chain reaction 心灵的裸舞心灵的裸舞-自传自传 My present researc
11、h work Degenerate PCR can be used to clone gene coding for enzyme 简并引物简并引物PCR-结合自己的研究工作结合自己的研究工作PCR application-example 1Codon:degenerateAnticodon:wobble密码子的简并性密码子的简并性Many amino acids are specified by more than one codon-degeneracy(简并性简并性).Codons specifying the same amino acid are called synonyms(同义
12、密码子同义密码子).GDegenerate primers(简并引物简并引物):an oligo pool derived from a protein sequence.His-Phe-Pro-Phe-Met-Lys can generate a primer 5-CAY TTY CCN TTY ATG AAR-3Y=Pyrimidine(C or T)N=any baseR=purine(A or G)Degenerate PCR(简并简并PCR)My research work:molecular biology research of laccase(漆酶漆酶)produced in
13、fungi -白腐真菌漆酶的分子生物学研究白腐真菌漆酶的分子生物学研究白腐真菌白腐真菌（白腐真菌（white rot fungi)白腐真菌是一白腐真菌是一类使木材呈白类使木材呈白色腐朽的丝状色腐朽的丝状真菌的总称真菌的总称（主要是担子（主要是担子菌）菌）已知的唯一能已知的唯一能在纯系培养中在纯系培养中有效地将木质有效地将木质素彻底降解为素彻底降解为CO2 和和H2O 的一类微生物的一类微生物木质素：以芳香族为基本结构的高度复杂聚合物木质素：以芳香族为基本结构的高度复杂聚合物 Science,2007,315(5813):804-807 对与木质素结构相似的异生物质污染物也对与木质素结构相似的异
14、生物质污染物也具有强大的降解能力具有强大的降解能力 木质素过氧化物酶（LiP）锰过氧化物酶（MnP）漆酶（漆酶（LacLac）白腐真菌木质素降解酶系白腐真菌木质素降解酶系非立体选择性和非特异性非立体选择性和非特异性 漆酶（漆酶（Laccase）含铜的多酚氧化含铜的多酚氧化酶酶,白腐真菌降解白腐真菌降解木质素的重要酶木质素的重要酶 具有广泛的底物具有广泛的底物作用范围和独特作用范围和独特的生物降解功能的生物降解功能 环境生物技术环境生物技术Biotechnology Advances 24(2006)500513 Use degenerate PCR(简并简并PCR)to clone lacca
15、se geneCopper-binding region is highly conservedCopper-binding region I:His-Trp-His-Gly-Phe-Phe-GlnCopper-binding region IV:His-Cys-His-Ile-Asp-Phe-His简并引物简并引物PCR获得获得1.6kb漆酶基因特异性序列漆酶基因特异性序列基因组步移技术获得基因组步移技术获得2118bp漆酶全长结构基因漆酶全长结构基因RACE和和RT-PCR克隆得到了克隆得到了1566bp 漆酶基因全长漆酶基因全长cDNA序列序列1 22kb1kb-我们自己的研究工作我们自
16、己的研究工作Question:By degenerate PCR,only the partial sequence of one gene can be obtained,How can we get the entire gene?Chromosome walking(染色体步移技术染色体步移技术)Long Distance inverse PCR Technique for Efficient Cloning of Flanking Sequences Adjacent to Known DNA Fragmentsinverse PCR(反向反向PCR)Reverse transcrip
17、tase(RT)-PCR逆转录逆转录PCR检测基因的转录量检测基因的转录量PCR application-example 2Reverse transcriptase(RT)-PCR逆转录逆转录PCRAAA(A)n5-CapmRNA(dT)1218 primeranneal5-CapAAA(A)n35Reverse transcriptiondNTP,RT5-CapAAA(A)n5cDNA:mRNA hybridRegularPCRRT-PCR application Clone cDNA of specific gene Detecting the expression level of g
18、ene Study this technique from experiment(实战中学习实战中学习)Detecting the expression level of gene by RT-PCRmRNAcDNART-PCR productRight panel:PCR amplification of the cloned lac1 cDNA using the cycle number obtainedfrom the left panelValidation of semiquantitative RT-PCR assays for expression level of lac1(
19、laccase gene)Left panel:Kinetics ofPCR amplification with the electrophoretic image shown at the top.The cycle number(28)that generates half maximal reaction wasused to analyse the expression of the gene.Eur.J.Biochem.(2004)271,318328RT-PCR analysis of transcription of lac1 induced by different conc
20、entrations of copperlac1:laccase genegpd:house-keeping geneThe expression levels were normalized by using the relative mRNA ratio(lac1/gpd)-semiquantitative RT-PCR 各种芳香化合物对各种芳香化合物对laccase 基因的转录调控效果是不同的基因的转录调控效果是不同的RT-PCR analysis of transcription patterns of lac1 induced by different aromatic compoundsveratric acidferulic acid(阿魏酸阿魏酸)2,5-xylidinevanillin芳香化合物芳香化合物Eur.J.Biochem.(2004)271,318328RT-PCR(反转录反转录PCR）:Effects of various copperconcentrations on lcc mRNAMeasure the laccase activity:Effects of various copper concentrations on laccase activity
