[學(xué)習(xí)]分子生物學(xué)楊洋第三章-dna的復(fù)制--基于dna復(fù)制原理的pcr技術(shù)

1、PCR（聚合酶鏈?zhǔn)椒磻?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

2、one end of the the DNA target sequence.,理論,,技術(shù),Denaturation (變性): The target DNA (template) is separated into two stands by heating to 95℃Primer annealing (退火): The temperature is reduced to around 55℃ to allow the pri

3、mers to anneal.Polymerization (elongation, extension) (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

4、 each PCR cycle.,,,,Extension (DNA polymerase),,InitialDNA,8,4,2,1,Number of DNA molecules (2n): 指數(shù)擴(kuò)增,Many cycles (25-35 in common) are performed to complete one PCR reaction, which resulted in an exponential amplifica

5、tion of the target DNA in which both 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 o

6、f template DNA, PCR can only be applied if some sequence information is known so that primers can be designed.,Figure 8-3 Substrates required for DNA synthesis,PCR Primers,Anneal on opposite strands of the target sequenc

7、e: 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-5°C),5’-ATTCCGATCGCTAATCGATGGC------

8、- TCCTGTGCA TTTCGCCACTAGAG-3’3’-TAAGGCTAGCGATTAGCTACCG-------AGGACACGTAAAGCGGTGATCTC-5’,5’-ATTCCGATCGCTAATCGATG-3’,3’-CACGTAAAGCGGTGATCTC-5’,forward primer,reverse primer,,,,5’-CTCTAGTGGCGAAATGCAC-3’,,,,Extension (DNA

9、polymerase),PCR Enzymes,Denaturation (變性): The target DNA (template) 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.Polyme

10、rization (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 thermophilic bacterium Thermus aquaticus (嗜

11、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(lái)的”,如果你的研究能力一般，那么，

12、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(jiǎng) ----- Kary Mullis (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(jī),PCR技術(shù)的發(fā)現(xiàn)充滿(mǎn)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(jìn)入塞特斯公司：與DNA合成相關(guān)的工作1983年4月：PCR的

13、最初想法高速公路的靈感230=10億想法付諸行動(dòng)1983年12月：第一個(gè)PCR反應(yīng)成功1984年塞特斯公司申請(qǐng)PCR技術(shù)專(zhuān)利,PCR技術(shù)引來(lái)的官司誰(shuí)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 the 1993 Nobel Prize in Chemistry for his invention of the polymeras

14、e chain reaction,心靈的裸舞-自傳,My present research workDegenerate PCR can be used to clone gene coding for enzyme 簡(jiǎn)并引物PCR----結(jié)合自己的研究工作,PCR application-example 1,Codon: degenerateAnticodon: wobble,密碼子的簡(jiǎn)并性,Many amino aci

15、ds are specified by more than one codon-degeneracy (簡(jiǎn)并性).Codons specifying the same amino acid are called synonyms (同義密碼子).,G,Degenerate primers (簡(jiǎn)并引物): an oligo pool derived from a protein sequence.His-Phe-Pro-Phe-

16、Met-Lys can generate a primer 5’-CAY TTY CCN TTY ATG AAR-3’Y= Pyrimidine (C or T)N= any baseR= purine (A or G),Degenerate PCR( 簡(jiǎn)并PCR),My research work: molecular biology research of laccase (漆酶) produced in fung

17、i --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(lèi)使木材呈白色腐朽的絲狀真菌的總稱(chēng)（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(lèi)微生物,,,,對(duì)與木質(zhì)素結(jié)構(gòu)相似的異生物質(zhì)污染物也具有強(qiáng)大的降解能力,木質(zhì)素過(guò)氧化物酶（LiP） 錳過(guò)氧化物酶（MnP）

18、 漆酶（Lac）,白腐真菌木質(zhì)素降解酶系,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(dú)特的生物降解功能環(huán)境生物技術(shù),Biotechnology Advances 24 (2006) 500–513,Use degenerate PCR( 簡(jiǎn)并PCR) to clone laccase gene,Copper-binding region is hi

19、ghly conserved,Copper-binding region I: His-Trp-His-Gly-Phe-Phe-Gln,Copper-binding region IV: His-Cys-His-Ile-Asp-Phe-His,簡(jiǎn)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(zhǎng)結(jié)構(gòu)基因,,RACE和RT-PCR克隆得到了1566bp 漆酶基因全長(zhǎng)cDNA序列,----我們自己的研究

20、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 Efficient Cloning of Flanking

21、 Sequences Adjacent to Known DNA Fragments,inverse PCR(反向PCR),Reverse transcriptase (RT)-PCR逆轉(zhuǎn)錄PCR檢測(cè)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)12~18 primer,anne

22、al,,,,5‘-Cap,AAA(A)n,,3‘,5‘,,Reverse transcription,dNTP, RT,,,,5‘-Cap,,AAA(A)n,,5‘,,cDNA:mRNA hybrid,,RegularPCR,RT-PCR application,Clone cDNA of specific geneDetecting the expression level of gene,Study this techniqu

23、e from experiment(實(shí)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 the cycle number obtainedfrom the left panel,Validation o

24、f semiquantitative RT-PCR assays for expression level of lac1(laccase gene),Left panel: Kinetics ofPCR amplification with the electrophoretic image shown at the top. The cycle number (28) that generates half maximal re

25、action wasused to analyse the expression of the gene.,Eur. J. Biochem. (2004) 271, 318–328,RT-PCR analysis of transcription of lac1 induced by different concentrations of copper,lac1: laccase genegpd: house-keeping gen

26、e,The expression levels were normalized by using the relative mRNA ratio (lac1/gpd)-semiquantitative RT-PCR,各種芳香化合物對(duì)laccase 基因的轉(zhuǎn)錄調(diào)控效果是不同的,Eur. J. Biochem. (2004) 271, 318–328,,RT-PCR(反轉(zhuǎn)錄PCR）:Effects of various copperc