Chapter4ManipulationofPurifiedDNA

1、1Chapter 4 Manipulation of Purified DNA2Contentsn4.1 The range of DNA manipulative enzymes 4.1.1 Nucleases 4.1.2 Ligases 4.1.3 Polymerases 4.1.4 DNA modifying enzymes 4.1.5 Topoisomerases3Contentsn4.2 Enzymes for cutting DNA-restriction endonucleases 4.2.1 The discovery and function of restriction e

2、ndonucleases 4.2.2 Type II restriction endonucleases cut DNA at specific nucleotide sequences 4.2.3 Blunt ends and sticky ends 4.2.4 The frequency of recognition sequences in a DNA molecule 4.2.5 Performing a restriction digest in the laboratory 4.2.6 Analysing the result of restriction endonuclease

3、 cleavage 4.2.7 Estimation of the sizes of DNA molecules 4.2.8 Mapping the positions of different restriction sites on a DNA molecule4Contentsn4.3 Ligation-joining DNA molecules together 4.3.1 The mode of action of DNA ligase 4.3.2 Sticky ends increase the efficiency of ligation 4.3.3 Putting sticky

4、 ends onto a blunt- ended molecule54.1 The range of DNA manipulative enzymesnFive classes based on the reaction types:(1).Nucleases (核酸酶):enzymes that cut, shorten or degrade nucleic acid molecules. (2).Ligases(連接酶): join nucleic acid molecules together.(3).Polymerases (聚合酶)make copies of molecules.

5、(4).Modifying enzymes(修飾酶) remove or add chemical groups.(5).Topoisomerases(拓撲異構(gòu)酶) introduce or remove supercoils from covalently closed-circular DNA.64.1 Nuclease(核酸酶)nNucleases degrade DNA molecules by breaking the phosphodiester bonds(磷酸二酯鍵) that link one nucleotide to the next in a DNA strand.nE

6、xonucleases remove nucleotides one at a time from the end of a DNA molecule.nEndonucleases :break internal phosphodiester bonds within a DNA molecule.7 Fig.4.1 The reactions catalysed by the two different kinds of nuclease. (a) an exonuclease,which removes nucleotides from the end of a DNA molecule.

7、 (b) an endonuclease, which breaks internal phosphodiester bond.8Exonuclease (外切核酸酶外切核酸酶)1. Bal31:npurified from the bacterium Alteromonas espejiana(交替單胞菌)nremove nucleotides from both strands of a double-stranded molecule.nThe longer time that Bal31 acting on a group of DNA molecules, the shorter r

8、esulting DNA fragments will be .n它能夠切開雙鏈DNA分子的兩條鏈. Bal31作用于DNA分子的時間越長,獲得的DNA片段就越短 9Exonuclease (外切核酸酶外切核酸酶)2. Exonuclease IIInFrom E.colinDegrade one strand of a double-stranded molecule, removes nucleotides only from the 3 terminus.n催化從dsDNA 3-OH逐一去除單核苷酸 n底物為dsDNA線狀或帶切口或缺口的環(huán)狀DNA，結(jié)果是在dsDNA上產(chǎn)生長的單鏈區(qū)。

9、n不降解單鏈DNA及3突出dsDNA 10Fig.4.2 The reactions catalysed by different types of exonuclease. (a) Bal31, which removes nucleotides from both strands of a double-stranded molecule. (b) exonuclease III, which removes nucleotides only from the 3 terminus.11Endonuclease (內(nèi)切核酸酶)1. S1(from the fungus Aspergillu

10、s oryzae-米曲霉): only cleaves single strands.n降解單鏈DNA或RNA，產(chǎn)生帶5磷酸的單核苷酸或寡核苷酸 n對dsDNA，dsRNA；DNA:RNA不敏感作用：用于分析DNA:RNA雜交體的結(jié)構(gòu) 去掉突出的單鏈尾，以產(chǎn)生平端 打開雙鏈cDNA合成中產(chǎn)生的發(fā)莢環(huán)12Endonuclease (內(nèi)切核酸酶)2. DNase I (deoxyribonuclease I),nprepared from cow pancreas (牛胰 ), ncut both single and double-stranded molecules.可作用單/雙鏈DNAnNo

11、n-specific sites it attack.切割無位點特異性 nRestriction endonucleases: cleave double-stranded DNA at specific recognition sites.13Fig.4.3 The reactions catalysed by different types of endonuclease. (a) S1 nuclease, which cleaves only single-stranded DNA, including single-stranded nicks in mainly double-str

12、anded molecules. (b) DNase I, which cleaves both single and double-stranded DNA. (c) A restriction endonuclease, which cleaves double-stranded DNA, but only at a limited number of sites.144.1.2 Ligase (連接酶)Functions（功能）: nto repair single-stranded breaks arising in double-stranded DNA during replica

13、tion.nJoin together two individual fragments of double-stranded DNA.15Fig 4.19 Ligation: the final step inConstruction of a recombinant DNA molecule164.1.3 Polymerase(聚合酶)nEnzymes that synthesize a new strand of DNA complementary to an existing DNA or RNA template.n催化DNA的合成(模板/引物/dNTP等)及其相輔的活性 Four

14、types of DNA polymereases usually used in genetic engineering:1) DNA polymerease I2) Klenow fragment3)Taq DNA polymerase4) Reverse transcriptase171. DNA polymerease I（DNA聚合酶I)nFrom E.coli (Ecoli DNA polymerase I，DNA pol I),單鏈多肽(109kDa), 三種活性1. 活性n 53DNA聚合酶活性n底物: 模板(ssDNA), 引物（帶3OH基） n 或 5突出DsDNA Mg2

15、+ dNTP DNApolyI1853外切核酸酶活性底物: dsDNA or DNA: RNA雜交體 活性：從5端降解dsDNA，也降解 RNA:DNA中的RNA(RNase H 活性) Mg2+ DNApolyI+ dNTP1935外切酶活性 底物：3-OH dsDNA or ssDNA Mg2+ DNApolyI活性：從3-OH端降解DNA，可被53聚合活性封閉。+ dNTP Mg2+ DNApolyI+ dNTPProofreading 20 Mg2+, 低限量DNase IdNTP DNApoly I產(chǎn)生切口（1）切口平移外切活性和合成活性共同作用使切口沿5-3方向平移若有放射性dNT

16、P, 則可標記成探針21 2.Klenow酶E.coli DNA polymerase I large fragment Klenow fragment在蛋白酶（枯草桿菌蛋白酶）裂解, 從全酶中除去53外切活性片段，而聚合活性和35外切活性不受影響，或基因工程而得 76kDa221. 活性共兩種, 同 DNApoly I聚合活性和35外切活性2.作用(1). 補平3凹端，注意要用足夠的 dNTP (2). 末端標記 A置換反應(yīng)， 被T4poly代替 B補平3-凹端的過程進行標記(3) cDNA克隆中合成第二鏈，僅利用DNA合成活性23 末端標記 （ 交換或置換反應(yīng)） Mg2+, Klenow

17、fragment -P32dATPA*T243、Taq DNA polymerase Taq DNA聚合酶：從棲熱水生菌（Thermus aquaticus)中分離純化的依賴DNA的DNA聚合酶。 63kDaActivities: 53 DNA polymerase 53exonuclease error rate of Taq DNA polymerase 特點：thermostable(耐高溫)。25依賴于RNA的DNA聚合酶，4、Reverse transcriptase Reverse transcriptase ( (反轉(zhuǎn)錄酶) 5 3合成DNA 無3 5外切活性1種類 兩種商品化的

18、反轉(zhuǎn)錄酶來自 AMV 禽成髓細胞瘤病毒 Mo-MLV(M-MuLV) Moloney鼠白血病病毒262. 活性 53DNA聚合活性（Mg+） RNase H 活性 273用途（1） cDNA克隆 （2） 5突出DNA的3端補平 （3） 測序反應(yīng) (代替 Klenow 大片段) 28Figure4.5 The reactions catalyzed by DNA polymerases (a) The basic reaction: a new DNA strand is synthesized in the 5 to 3direction (b) DNA polymerase , which

19、initially fills in nicks but then continues to synthesize a new strand, degrading the exciting one as it process. (c) The Klenow fragment, which only fills in nicks. (d) Reverse transcriptase, which uses a template of RNA294.1.4 DNA modifying enzymes1) Alkaline phosphatase (堿性磷酸酶): nfrom E.coli, cal

20、f intestinal tissue or arctic shrimp 牛小腸堿性磷酸酶（CIP，CIAP） 細菌堿性磷酸酶（BAP） 蝦的堿性磷酸酶 (SAP)nRemoves the phosphate group present at the 5terminus of a DNA molecule.去除DNA分子5末端的磷酸基團n用途：主要用于去除載體DNA 5磷酸,防止DNA 片段自身環(huán)狀，提高重組DNA檢出率。 以及標記前（5端）除5磷酸304.1.4 DNA modifying enzymes2) Polynucleotide kinase (多核苷酸激酶)nFrom E.col

21、i infected with T4 phagenAdd phosphate groups onto free 5 terminus. 在 DNA分子5游離末端添加磷酸基團314.1.4 DNA modifying enzymes3) Terminal deoxynucleotidyl transferase(末端轉(zhuǎn)脫氧核苷酰酶,末端轉(zhuǎn)移酶):nFrom calf thymus (胸腺)tissuenAdds one or more deoxyribonucleotides ( 脫氧核糖核苷酸) onto the 3terminus of DNA moleculen催化脫氧核苷酸添加到DNA分

22、子的3-OH末端，催化作用不需要模板。n底物：具有3-OH末端的單鏈DNA,也可以是具有3-OH突出末端的雙鏈DNA.n用途：分別給外源DNA片段及載體DNA加上互補的同聚物尾巴，使它們可以重組起來。 32334.1.5 Topoisomerase(拓撲異構(gòu)酶)nTo change the conformation of covalently closed-circular DNA (e.g. plasmid DNA) by introducing or removing supercoils.344.2 Enzymes for cutting DNA-restriction endonucl

23、eases(限制性內(nèi)切酶)nGene cloning requires DNA molecules being cut in a precise and reproducible fashion.基因克隆要求DNA被準確切割且可重復(fù)(1) Each vector must be cleaved at a single position; and be cut at exactly the same position on the circular. 載體在單一位置切割；載體在特定位點切割nDNA to be cloned often need to be cleaved(1) The desi

24、red gene usually have a size of only 2-3Kb.(2) Most vectors exhibit a preference DNA size that will be inserted. 35nFig 4.7 The need for very precise cutting manipulations in a gene cloning experiment.36nRestriction endonucleases allow DNA to be cut in the precise, reproducible manner.nThe discovery

25、 of restriction endonucleases was one of the breakthroughs in the development of genetic engineering.nW. Arber, H Smith, D. Nathans, 1978, Nobel Prizes374.2.1 The discovery and function of restriction endonucleasesnRestriction and modification (限制與修飾)nIn the early 1950s, host-controlled restriction(

26、宿主控制性限制): a phenomenon shown that some strains of bacteria are immune to bacteriophage infection.381. Phenomenonnpfu Plaque forming unitnEOP Efficiency of platingE.coli KE.coli BEOP=10-4EOP=1EOP=1EOP=10-439n說明K和B菌株中存在一種限制系統(tǒng) 可排除外來的DNA n10-4的存活率是由宿主修飾系統(tǒng)作用的結(jié)果 n限制作用：指一定類型的細菌可以通過限制酶的作用，破壞入侵的噬菌體DNA，導(dǎo)致噬菌體的

27、寄主幅度受到限制。n修飾作用:寄主本身的DNA，由于合成后通過甲基化酶的作用得以甲基化 ，使DNA得以修飾，從而免遭自身限制性酶的破壞。n限制與修飾的作用：保護自身的DNA不受限制；破壞外源DNA使之迅速降解。40Fig4.8 The function of a restriction endonuclease in a bacterial cell: (a) phage DNA is cleaved, but (b) bacterial DNA is not41nSo far, over 3000 different restriction endonucleases have been c

28、haracterized.nhttp:/ I, II and III type I (1%) 種類少 如 EcoK EcoB type II 98% typeIII (6 Not I GCGGCCGC 8 BbvCI CCTCAGC 7 PpuMI RGGWCCY 7R=A or G Y=C or T M=A or C K=G or T S=C or G W=A or T H=A or C or T B=C or G or T V=A or C or G D=A or G or T N=A or C or G or T4748Table 3.3 Some restriction endonuc

29、leases and their recognition sitesEnzymeRecognition sequences4-base cuttersMboI, DpnI, Sau3AI/GATCMspI, HpaIIC/CGGAluIAG/CTHaeIIIGG/CCTaiIACGT/6-Base cuttersBglIIA/GATCTClaIAT/CGATPvuIICAG/CTGPvuICGAT/CGKpnIGGTAC/C8-Base cuttersNotIGC/GGCCGCSbfICCTGCA/GG494.2.3 Blunt ends(平端) and sticky ends(粘端)n大多數(shù)

30、限制性內(nèi)切酶的識別序列是一個回文對稱順序(palindrome )，即有一個中心對稱軸，從這個軸朝二個方向“讀”都完全相同。這種酶的切割可以有兩種方式。nEcoR In5GAA | TTC3n 3CTT | AAG5Axis of symmetry50(1) blunt end/ flush end在同一位置上切割雙鏈，產(chǎn)生平頭末端。例如HaeIII的識別位置是： 5GGCC33CCGG551(2) Sticky end另一是交錯切割，結(jié)果形成兩條單鏈末端，這種末端的核苷酸順序是互補的，可形成氫鍵，所以稱為粘性末端。如EcoRI的識別順序為：5GAA | TTC3 3CTT | AAG552D

31、NA joined by sticky ends53n(3) restriction endonucleases with different recognition sequences may produce the same sticky ends. (同尾酶: isocaudamer)：具有不同識別位點的限制性內(nèi)切酶能夠產(chǎn)生相同的粘末端。nBam HI G/GATCC CCTAG/G Bal I T/GATCA ACTAG/T54nIsoschizomers(同裂酶): restriction enzymes with the same sequence specificity and

32、cut site. e.g.HindII HincII GTY/RAC MobI Sau3AI /GATCnneoschizomers (新裂酶): enzymes that recognize the same sequence but cleave at different points.nSmaI (CCC/GGG) and XmaI (C/CCGGG)55Fig.4.9 The ends produced by cleavage of DNA with different restriction endonucleases. 56Fig 3.3 The generation of th

33、ree new restriction sites after filling in the overhangs produced byendonuclease EcoRI and ligating the products together.574.2.4 The frequency of recognition sequences in a DNA moleculeDNA分子中識別序列的出現(xiàn)頻率nThe number and size of the fragments generated by a restriction enzyme depend on the frequency of

34、occurrence of the target site in the DNA to be cut.nAssumption: 50% GC contents and random distribution of a four bases, na four base recognition site (e.g. GATC) occurs every , N=44 (256)bpnA hexanucleotide (e.g. GGATCC) sequence: N=46=4096 bp58Fig.4.10 Restriction of the DNA molecule. The numbers

35、are the fragment sizes in base pairs.59Table3.4 Average fragment size (bp) produced by different enzymes with DNA from different sourcesEnzymeTargetArabodopsis擬南芥Nematode線蟲Drosophila果蠅E.coliHumanApaIGGGCCC25 00040 0006 00015 0002 000AvrIICCTAGG15 00020 00020 000150 0008 000BamHIGGATCC6 0009 0004 000

36、5 0005 000DraITTTAAA2 0001 0001 0002 0002 000Spe IACTAGT8 0008 0009 00060 00010 00060nIn practice, restriction sites are not evenly spaced out along a DNA molecule.nAnd in a DNA molecule the nucleotides are not randomly ordered.614.2.5 Performing a restriction digest in the laboratory限制性酶切實驗Fig.4.11

37、 Performing a restriction digest in the laboratory.Incubate at 70C or add EDTAto stop the reaction;62nDithiothreitol (DTT, 二硫蘇糖醇): reducing agent, stabilizes the enzyme and prevents its inactivation. 63nProviding the right conditions is very important for the restriction endonuclease:nIncorrect NaCl

38、 or Mg2+ concentration may not only decrease the enzyme activity, but may also cause changes in the specificity of the enzyme, so that DNA cleavage occurs at additional, non-standard recognition sequences. (星號or 星星活性)64Star activity (星號活性)nUnder extreme conditions, such as elevated pH or low ionic s

39、trength, restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. The altered specificity is known as star activity.n EcoRI* (EcoRI star activity): N/AATTC65引起星星活性的因素n 高濃度甘油（5%） n 酶過量（100U/g） n 低離子強度（pH8.0)n 有機溶劑 PMSD(二甲基亞

40、砜)、乙醇、乙二醇、二甲基乙酰胺（dimethylacetamide）、二甲基甲酰胺（dimethylformamide）n 用其它二價陽離子代替Mg+, nMn+，Cu+，Co+, Zn+ 66抑制星星活性的條件（措施） 減少酶的用量，避免過量酶切,減少甘油濃度 保證反應(yīng)體系中無有機溶劑（乙醇等） 提高離子強度到100150mM (如果不會抑制的話) 降低反應(yīng)pH至pH7.0 使用Mg+作為二價陽離子 674.2.6 Analyzing the result of restriction endonuclease cleavage對限制性內(nèi)切酶的酶切結(jié)果分析 nA DNA molecule

41、is cut or not can be determined easily by testing the viscosity of the solution: larger DNA molecules result in a more viscous solution than smaller ones, so cleavage is associated with a decrease in viscosity（粘度）.nThe number and sizes of the individual cleavage products was determined by gel electr

42、ophoresis（凝膠電泳）. 68Separation of molecules by gel electrophoresis-通過凝膠電泳分離生物大分子nDNA: negative chargednDNA cannot be separated by standard electrophoresis because of the same shape and similar charge-to-mass ratio (荷質(zhì)比).nDNA can be separated according to their size by gel electrophoresis.nIn practice

43、, the composition of the gel determines the size of the DNA molecules that can be separated.69Fig.4.12 (a) standard electrophoresis does not separate DNA fragments of different sizes, whereas (b) gel electrophoresis does.70Visualizing DNA molecules in a gel使凝膠中的DNA分子可視化1. Staining : Ethidium bromide

44、 (EtBr-)nDNA fragment are clearly visible under ultra-violet irradiation after EtBr staining.Fig.4.13 Visualizing DNA bands in anagarose gel by EtBr staining and Ultraviolet (UV) irradiation. 71Autoradiography (放射自顯影) of radioactively labelled DNAnMore sensitive than the EtBr staining methodnThe DNA

45、 is usually labelled before electrophoresis by incorporating of a radioactive marker(放射性標記) into the individual molecules, the DNA can be visualized by placing an X-ray-sensitive photographic film over the gel.72Fig 4.14 The use of autoradiography to visualize radioactively labelled DNA in an agaros

46、e gel.73nNick translation(缺口平移)nEnd filling (末端填平)Fig. 4.15 Radioactive labeling: (a) structure of -32p- ATP, (b) labelling DNA by nick translation, and (c) labelling DNA by end filling 744.2.7 Estimation of the sizes of DNA molecules (DNA分子大小的估計）nD=a-b(logM)nD: the distance moved, M: the molecular

47、weight, a and b are constants that depend on the electrophoresis conditions.75Fig.4.16 Estimation of the sizes of DNA fragments in an agarose gel.764.2.8 Mapping the positions of different restriction sites on a DNA molecule 通過作圖標出一個DNA分子上的不同限制性位點nRestriction map(限制性酶切圖譜): a map showing the position

48、s of different restriction sites in a DNA molecule.n指一系列限制酶的特異識別序列在DNA鏈上的出現(xiàn)頻率和它們之間的相對位置。77Fig 4.17 using a restriction map to work out which restriction endonucleases should be used to obtain DNA fragments containing individual genes.78nDouble digestion(雙酶切): cleavage of a DNA molecule with two diff

49、erent restriction endonucleases, either concurrently or consecutively.nPartial digestion (部分酶切): treatment of a DNA molecule with a restriction endonuclease under such conditions that only a fraction of all the recognition sites are cleaved.79Fig 4.18 Restriction mapping. This example shows how the

50、positions of the Xbal, Xhol and Kpnl sites on the DNA molecule can be determined.804.3 Ligation joining DNA molecules togetherFig 4.19 Ligation: the final step inConstruction of a recombinant DNA molecule814.3.1 The mode of action of DNA ligasenT4 DNA ligase:催化DNA 5磷酸基與3羥基之間形成磷酸二酯鍵Fig 4.20 The diffe

51、rent joining reactionsCatalysed by DNA ligase: (a) ligation ofBlunt-ended molecules, and (b) ligationOf sticky-ended molecules.82nT4 DNA ligase: require ATPnE.coli DNA ligase: NAD+83Fig 3.6 Action of DNA ligase84Three methods for joining DNA fragmentsnDNA ligase: to join covalently the annealed cohe

52、sive endsnT4 DNA ligase to catalyse between blunt-ended fragmentsnTerminal deoxynucleotidyltransferase(末端轉(zhuǎn)移酶): to synthesize homopolymeric (同聚的) 3 single-stranded tails.85Fig 3.7 use of DNAligase to created a covalent DNA recombinant joinedthrough associationof termini generated by EcoRI.86Ligation

53、reaction(連接反應(yīng))nLigation temperature: 4 15Cnligation reaction to favor the formation of recombinants:1. High DNA concentration2. Treat linearized plasmid vector DNA with alkaline phosphatase: to remove 5-terminal phosphate groups. 87Fig 3.8 Application of alkaline phosphatase treatment to preventreci

54、rcularization of vector plasmid without insertion of foreign DNA884.3.2 Sticky ends increase the efficiency of ligation -粘末端增加連接的效率nBlunt end ligation（平端連接）: low efficiencynSticky end ligation（粘端連接）: high efficiency.89Fig 4.20 The different joining reactionsCatalysed by DNA ligase: (a) ligation ofBl

55、unt-ended molecules, and (b) ligationOf sticky-ended molecules.904.3.3 Putting sticky ends onto a blunt-ended moleculenLinker (DNA接頭): A synthetic, double-stranded oligonucleotide used to attach sticky ends to a blunt-ended molecule. It is blunt-ended, but contains a restriction site.91Fig.4.21 Link

56、ers and their use: (a) the structure of a typical linker, and (b) the attachment of linkers to a blunt-ended molecule.Fig.4.22 A possible problem with the use of linkers.9293Fig.4.23 Adaptors and the potential problem with their use.94Fig.4.24 The distinction between the 5 and 3 temini of a polynucl

57、eotide.95Figure4.25 The use of adaptors :(a) The actual structure of an adaptor, showing the modified 5-OH terminus 96Fig. 4.25 The use of adaptors: (a) The actual structure of an adaptor, showing the modified 5-OH terminus. (b) Conversion of blunt ends to sticky ends through the attachment of adapt

58、ors.97(3) Producing sticky ends by homopolymer tailingnHomopolymer tailing ( 同聚物加尾): the attachment of a sequence of identical nucleotides (e.g. AAAAA) to the end of a nucleic acid molecule, usually referring to the synthesis of single-stranded homopolymer extensions on the ends of a double-stranded

59、 DNA molecule.98Fig.4.26 Homopolymer tailing: (a) synthesis of a homopolymer tail, (b) construction of a recombinant DNA molecule from a tailed vector plus tailed insert DNA, and (c) repair of the recombinant DNA molecule. dCTP=2-deoxycytidine 5-triphosphate.99本章小結(jié)n4.1 DNA操作酶的范圍The range of DNA mani

60、pulative enzymes 4.1.1 核酸酶Nucleases 基本概念, 外切核酸酶, 代表酶Bal31, Exonuclease III; 內(nèi)切核酸酶, S1, DNase I 4.1.2 連接酶Ligases 概念, 功能 4.1.3 聚合酶Polymerases 概念, 幾種常用聚合酶(DNApol I, klenow酶, Taq DNA 聚合酶, 反轉(zhuǎn)錄酶)的特性,典型應(yīng)用 4.1.4 DNA 修飾酶 DNA modifying enzymes幾種常用DNA 修飾酶(堿性磷酸酶,T4多核苷酸激酶,末端轉(zhuǎn)移酶) 的特性,主要用途 4.1.5 拓撲異構(gòu)酶Topoisomerase