1、PCR（聚合酶鏈式反應(yīng)）,Polymerase chain reaction -An important technique based on 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.,理論,技術(shù),Denaturation (變性): The target DNA (t

2、emplate) is separated into two stands by heating to 95 Primer annealing (退火): The temperature is reduced to around 55 to allow the primers to anneal. Polymerization (elongation, extension) (延伸): The temperature is increased to 72 for optimal polymerization step which uses dNTPs and requires Mg+.,The

3、 principle of PCR: Three different steps proceed in each PCR cycle.,Extension (DNA polymerase),Initial DNA,8,4,2,1,Number of DNA molecules (2n): 指數(shù)擴增,Many cycles (25-35 in common) are performed to complete one PCR reaction, which resulted in an exponential amplification of the target DNA in which bo

4、th forward and reverse primers pair.,DNA template (模板),Any source of DNA 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 some sequence information is known so that primers can be designed.,Figu

5、re 8-3 Substrates required for DNA synthesis,PCR Primers,Anneal on opposite strands of the target sequence: forward and reverse primers （正向引物和反向引物） Have similar G+C contents (Tm) so that they anneal to their complementary sequences at similar temperatures (anneal temperature: Tm-5C),5-ATTCCGATCGCTAA

6、TCGATGGC- TCCTGTGCA TTTCGCCACTAGAG-3 3-TAAGGCTAGCGATTAGCTACCG-AGGACACGTAAAGCGGTGATCTC-5,5-ATTCCGATCGCTAATCGATG-3,3-CACGTAAAGCGGTGATCTC-5,forward primer,reverse primer,5-CTCTAGTGGCGAAATGCAC-3,Extension (DNA polymerase),PCR Enzymes,Denaturation (變性): The target DNA (template) is separated into two sta

7、nds by heating to 95 Primer annealing (退火): The temperature is reduced to around 55 to allow the primers to anneal. Polymerization (elongation, extension) (延伸): The temperature is increased to 72 for optimal polymerization step which uses dNTPs and requires Mg2+.,Taq polymerase ：isolated from the th

8、ermophilic bacterium Thermus aquaticus (嗜熱菌）, 耐熱DNA聚合酶，耐高溫 It has no 3 to 5 proofreading exonuclease activity High-accuracy DNA polymerase is available commercially(高保真酶）,PCR儀,Discovery of PCR technique,希望大家不僅僅知道“是什么”，而且也了解“為什么”和“結(jié)論是怎么得出來的”,如果你的研究能力一般，那么，改革本研究領(lǐng)域中大家習(xí)以為常的最基本的技術(shù)，你就有可能獲諾貝爾獎 - Kary Mulli

9、s (卡里.穆利斯),The replication of DNA,哥倫布豎立雞蛋的故事 DNA分子的拷貝術(shù)，復(fù)印機,PCR技術(shù)的發(fā)現(xiàn)充滿傳奇色彩 Kary Mullis (卡里.穆利斯) 1972年獲得加州大學(xué)伯克利分校生物化學(xué)博士學(xué)位 1979年進入塞特斯公司：與DNA合成相關(guān)的工作 1983年4月：PCR的最初想法 高速公路的靈感 230=10億 想法付諸行動 1983年12月：第一個PCR反應(yīng)成功 1984年塞特斯公司申請PCR技術(shù)專利,PCR技術(shù)引來的官司 誰發(fā)明了PCR？DNA聚合酶的發(fā)現(xiàn)者？ 紙上談兵 Kary Mullis (卡里.穆利斯)勝訴,Kary Mullis won

10、the 1993 Nobel Prize in Chemistry for his invention of the polymerase chain reaction,心靈的裸舞-自傳,My present research work Degenerate PCR can be used to clone gene coding for enzyme 簡并引物PCR -結(jié)合自己的研究工作,PCR application-example 1,Codon: degenerate Anticodon: wobble,密碼子的簡并性,Many amino acids are specified by

11、 more than one codon-degeneracy (簡并性). Codons specifying the same amino acid are called synonyms (同義密碼子).,G,Degenerate primers (簡并引物): 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-3 Y= Pyrimidine (C or T) N= any base R= purine

12、 (A or G),Degenerate PCR( 簡并PCR),My research work: molecular biology research of laccase (漆酶) produced in fungi -白腐真菌漆酶的分子生物學(xué)研究,白腐真菌,白腐真菌（white rot fungi),白腐真菌是一類使木材呈白色腐朽的絲狀真菌的總稱（主要是擔(dān)子菌） 已知的唯一能在純系培養(yǎng)中有效地將木質(zhì)素徹底降解為CO2 和H2O 的一類微生物,對與木質(zhì)素結(jié)構(gòu)相似的異生物質(zhì)污染物也具有強大的降解能力,木質(zhì)素過氧化物酶（LiP） 錳過氧化物酶（MnP） 漆酶（Lac）,白腐真菌木質(zhì)素降解酶系

13、,非立體選擇性和非特異性,漆酶（Laccase）,含銅的多酚氧化酶,白腐真菌降解木質(zhì)素的重要酶 具有廣泛的底物作用范圍和獨特的生物降解功能 環(huán)境生物技術(shù),Biotechnology Advances 24 (2006) 500513,Use degenerate PCR( 簡并PCR) to clone laccase gene,Copper-binding region is highly conserved,Copper-binding region I: His-Trp-His-Gly-Phe-Phe-Gln,Copper-binding region IV: His-Cys-His-I

14、le-Asp-Phe-His,簡并引物PCR獲得1.6kb漆酶基因特異性序列,基因組步移技術(shù)獲得2118bp漆酶全長結(jié)構(gòu)基因,RACE和RT-PCR克隆得到了 1566bp 漆酶基因全長cDNA序列,-我們自己的研究工作,Question:,By degenerate PCR, only the partial sequence of one gene can be obtained, How can we get the entire gene?,Chromosome walking (染色體步移技術(shù)),Long Distance inverse PCR Technique for Effi

15、cient Cloning of Flanking Sequences Adjacent to Known DNA Fragments,inverse PCR (反向PCR),Reverse transcriptase (RT)-PCR 逆轉(zhuǎn)錄PCR檢測基因的轉(zhuǎn)錄量,PCR application-example 2,Reverse transcriptase (RT)-PCR 逆轉(zhuǎn)錄PCR,AAA(A)n,5-Cap,mRNA,(dT)1218 primer,anneal,5-Cap,AAA(A)n,3,5,Reverse transcription,dNTP, RT,5-Cap,AAA(A

16、)n,5,cDNA:mRNA hybrid,Regular PCR,RT-PCR application,Clone cDNA of specific gene Detecting the expression level of gene,Study this technique from experiment(實戰(zhàn)中學(xué)習(xí)),Detecting the expression level of gene by RT-PCR,mRNA,cDNA,RT-PCR product,Right panel: PCR amplification of the cloned lac1 cDNA using t

17、he cycle number obtained from the left panel,Validation of semiquantitative RT-PCR assays for expression level of lac1(laccase gene),Left panel: Kinetics of PCR amplification with the electrophoretic image shown at the top. The cycle number (28) that generates half maximal reaction was used to analyse the expression of the gene.,Eur. J. Biochem. (2004) 271, 318328,RT-PCR analysis of transcription of lac1 induced by different concentrations of copper,lac1: laccase gene gpd: house-keeping gene,The expression levels were normalized by using the