Chapter 4 Manipulation of Purified DNA

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(连接酶): join nucleic acid molecules together.(3).Polymerases (聚合酶)make copies of molecules.

5、(4).Modifying enzymes(修饰酶) remove or add chemical groups.(5).Topoisomerases(拓扑异构酶) introduce or remove supercoils from covalently closed-circular DNA.64.1 Nuclease(核酸酶)nNucleases degrade DNA molecules by breaking the phosphodiester bonds(磷酸二酯键) 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(交替单胞菌)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它能够切开双链DNA分子的两条链. Bal31作用于DNA分子的时间越长,获得的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催化从dsDNA 3-OH逐一去除单核苷酸 n底物为dsDNA线状或带切口或缺口的环状DNA，结果是在dsDNA上产生长的单链区。

9、n不降解单链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 (内切核酸酶)1. S1(from the fungus Aspergillu

10、s oryzae-米曲霉): only cleaves single strands.n降解单链DNA或RNA，产生带5磷酸的单核苷酸或寡核苷酸 n对dsDNA，dsRNA；DNA:RNA不敏感作用：用于分析DNA:RNA杂交体的结构 去掉突出的单链尾，以产生平端 打开双链cDNA合成中产生的发荚环12Endonuclease (内切核酸酶)2. DNase I (deoxyribonuclease I),nprepared from cow pancreas (牛胰 ), ncut both single and double-stranded molecules.可作用单/双链DNAnNo

11、n-specific sites it attack.切割无位点特异性 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 (连接酶)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)及其相辅的活性 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),单链多肽(109kDa), 三种活性1. 活性n 53DNA聚合酶活性n底物: 模板(ssDNA), 引物（带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杂交体 活性：从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：从3-OH端降解DNA，可被53聚合活性封闭。+ dNTP Mg2+ DNApolyI+ dNTPProofreading 20 Mg2+, 低限量DNase IdNTP DNApoly I产生切口（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, 则可标记成探针21 2.Klenow酶E.coli DNA polymerase I large fragment Klenow fragment在蛋白酶（枯草杆菌蛋白酶）裂解, 从全酶中除去53外切活性片段，而聚合活性和35外切活性不受影响，或基因工程而得 76kDa221. 活性共两种, 同 DNApoly I聚合活性和35外切活性2.作用(1). 补平3凹端，注意要用足够的 dNTP (2). 末端标记 A置换反应， 被T4poly代替 B补平3-凹端的过程进行标记(3) cDNA克隆中合成第二链，仅利用DNA合成活性23 末端标记 （ 交换或置换反应） Mg2+, Klenow

17、fragment -P32dATPA*T243、Taq DNA polymerase Taq DNA聚合酶：从栖热水生菌（Thermus aquaticus)中分离纯化的依赖DNA的DNA聚合酶。 63kDaActivities: 53 DNA polymerase 53exonuclease error rate of Taq DNA polymerase 特点：thermostable(耐高温)。25依赖于RNA的DNA聚合酶，4、Reverse transcriptase Reverse transcriptase ( (反转录酶) 5 3合成DNA 无3 5外切活性1种类 两种商品化的

18、反转录酶来自 AMV 禽成髓细胞瘤病毒 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端补平 （3） 测序反应 (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 (碱性磷酸酶): nfrom E.coli, cal

20、f intestinal tissue or arctic shrimp 牛小肠碱性磷酸酶（CIP，CIAP） 细菌碱性磷酸酶（BAP） 虾的碱性磷酸酶 (SAP)nRemoves the phosphate group present at the 5terminus of a DNA molecule.去除DNA分子5末端的磷酸基团n用途：主要用于去除载体DNA 5磷酸,防止DNA 片段自身环状，提高重组DNA检出率。 以及标记前（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游离末端添加磷酸基团314.1.4 DNA modifying enzymes3) Terminal deoxynucleotidyl transferase(末端转脱氧核苷酰酶,末端转移酶):nFrom calf thymus (胸腺)tissuenAdds one or more deoxyribonucleotides ( 脱氧核糖核苷酸) onto the 3terminus of DNA moleculen催化脱氧核苷酸添加到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末端的单链DNA,也可以是具有3-OH突出末端的双链DNA.n用途：分别给外源DNA片段及载体DNA加上互补的同聚物尾巴，使它们可以重组起来。 32334.1.5 Topoisomerase(拓扑异构酶)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(限制性内切酶)nGene cloning requires DNA molecules being cut in a precise and reproducible fashion.基因克隆要求DNA被准确切割且可重复(1) Each vector must be cleaved at a single position; and be cut at exactly the same position on the circular. 载体在单一位置切割；载体在特定位点切割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 (限制与修饰)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说明K和B菌株中存在一种限制系统 可排除外来的DNA n10-4的存活率是由宿主修饰系统作用的结果 n限制作用：指一定类型的细菌可以通过限制酶的作用，破坏入侵的噬菌体DNA，导致噬菌体的

27、寄主幅度受到限制。n修饰作用:寄主本身的DNA，由于合成后通过甲基化酶的作用得以甲基化 ，使DNA得以修饰，从而免遭自身限制性酶的破坏。n限制与修饰的作用：保护自身的DNA不受限制；破坏外源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%) 种类少 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大多数

30、限制性内切酶的识别序列是一个回文对称顺序(palindrome )，即有一个中心对称轴，从这个轴朝二个方向“读”都完全相同。这种酶的切割可以有两种方式。nEcoR In5GAA | TTC3n 3CTT | AAG5Axis of symmetry50(1) blunt end/ flush end在同一位置上切割双链，产生平头末端。例如HaeIII的识别位置是： 5GGCC33CCGG551(2) Sticky end另一是交错切割，结果形成两条单链末端，这种末端的核苷酸顺序是互补的，可形成氢键，所以称为粘性末端。如EcoRI的识别顺序为：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)：具有不同识别位点的限制性内切酶能够产生相同的粘末端。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分子中识别序列的出现频率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拟南芥Nematode线虫Drosophila果蝇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限制性酶切实验Fig.4.11

37、 Performing a restriction digest in the laboratory.Incubate at 70C or add EDTAto stop the reaction;62nDithiothreitol (DTT, 二硫苏糖醇): 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. (星号or 星星活性)64Star activity (星号活性)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 高浓度甘油（5%） n 酶过量（100U/g） n 低离子强度（pH8.0)n 有机溶剂 PMSD(二甲基亚

40、砜)、乙醇、乙二醇、二甲基乙酰胺（dimethylacetamide）、二甲基甲酰胺（dimethylformamide）n 用其它二价阳离子代替Mg+, nMn+，Cu+，Co+, Zn+ 66抑制星星活性的条件（措施） 减少酶的用量，避免过量酶切,减少甘油浓度 保证反应体系中无有机溶剂（乙醇等） 提高离子强度到100150mM (如果不会抑制的话) 降低反应pH至pH7.0 使用Mg+作为二价阳离子 674.2.6 Analyzing the result of restriction endonuclease cleavage对限制性内切酶的酶切结果分析 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（凝胶电泳）. 68Separation of molecules by gel electrophoresis-通过凝胶电泳分离生物大分子nDNA: negative chargednDNA cannot be separated by standard electrophoresis because of the same shape and similar charge-to-mass ratio (荷质比).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使凝胶中的DNA分子可视化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 (放射自显影) of radioactively labelled DNAnMore sensitive than the EtBr staining methodnThe DNA

45、 is usually labelled before electrophoresis by incorporating of a radioactive marker(放射性标记) 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分子大小的估计）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 通过作图标出一个DNA分子上的不同限制性位点nRestriction map(限制性酶切图谱): 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异识别序列在DNA链上的出现频率和它们之间的相对位置。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(双酶切): 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磷酸基与3羟基之间形成磷酸二酯键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(末端转移酶): 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(连接反应)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 -粘末端增加连接的效率nBlunt end ligation（平端连接）: low efficiencynSticky end ligation（粘端连接）: 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 molecule(1)Linker (DNA接头): 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 Li

56、nkers 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 polynu

57、cleotide.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 ada

58、ptors.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-strand

59、ed 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本章小结n4.1 DNA操作酶的范围The range of DNA ma

60、nipulative enzymes 4.1.1 核酸酶Nucleases 基本概念, 外切核酸酶, 代表酶Bal31, Exonuclease III; 内切核酸酶, S1, DNase I 4.1.2 连接酶Ligases 概念, 功能 4.1.3 聚合酶Polymerases 概念, 几种常用聚合酶(DNApol I, klenow酶, Taq DNA 聚合酶, 反转录酶)的特性,典型应用 4.1.4 DNA 修饰酶 DNA modifying enzymes几种常用DNA 修饰酶(碱性磷酸酶,T4多核苷酸激酶,末端转移酶) 的特性,主要用途 4.1.5 拓扑异构酶Topoisomera