分子生物学教学课件：CHAPTER 20 Techniques of Molecular Biology

1、1nMolecular Biology2知识准备：知识准备：1 1活细胞的复杂性活细胞的复杂性 （细胞结构、细胞器、细胞功能（细胞结构、细胞器、细胞功能-）2 2生物大分子结构与功能（核酸、蛋白质、多糖、脂质生物大分子结构与功能（核酸、蛋白质、多糖、脂质-）3 3计算机计算机 （程序、软件、生物信息学）（程序、软件、生物信息学）基因的分子生物学基因的分子生物学n1 1分子生物学实验方法分子生物学实验方法n2 2模式生物模式生物n3 3比较基因组学和动物多样性进化比较基因组学和动物多样性进化 3基本知识基本知识: : 本课程主要从生物大分子的水平来阐述从DNA到RNA到蛋白质的基因表达的重要生命

2、过程。语言能力语言能力: : 重点重点提高提高使用英文理解分子生物学的能力使用英文理解分子生物学的能力，使大家能够较轻松的阅读分子生物学方面的文献，获得最新的知识和发展方向。Objectives of the Course :4Introduction of textbook5Introduction of author of textbooknborn April 6, 1928nHe studied at the University of Chicago and Indiana University nbest known as one of the co-discoverers of

3、the structure of DNA. Watson, Francis Crick, and Maurice Wilkins were awarded the 1962 Nobel Prize in Physiology or Medicine James D. Watson as Director of Cold Spring Harbor Laboratory research emphasis to the study of cancer. Between 1988 and 1992 he was associated with the National Institutes of

4、Health, helping to establish the Human Genome Project. He has written many science books, including the seminal textbook The Molecular Biology of the Gene (1965) and his bestselling book The Double Helix (1968) about the DNA Structure discovery.6分子生物学常用参考书目分子生物学常用参考书目参考书参考书1 Molecular Biology by Rob

5、ert F.Weaver（第二版影印版）科学出版社科学出版社2Gene VIII by Benjamin Lewin （影印版）科学出版社科学出版社3Genome by Terry Brown 科学出版社7分子生物学实验参考书目分子生物学实验参考书目实验参考书实验参考书 1 . 分子克隆实验指南分子克隆实验指南 第三版第三版 译者: 黄培堂 科学出版社2. 精编分子生物学实验指南精编分子生物学实验指南译者: 颜子颖 科学出版社科学出版社3. 现代分子生物学实验技术现代分子生物学实验技术卢圣栋教授主编 中国协和医科大学出版社8n期末考试（期末考试（80）：）：对知识掌握和综合运对知识掌握和综合运

6、用能力的考察用能力的考察n平时小考（平时小考（10）：养成平时学习的好习：养成平时学习的好习惯，共惯，共2-3次。次。n出席情况（出席情况（10）：如果有如果有3次以上的缺次以上的缺席记录，席记录， 该分值为零。该分值为零。9Molecular biology is the attempt to understand biological phenomena in molecular term (broadly)Molecular biology is the study of gene structure and function at the molecular level, includ

7、ing DNA replication, transcription, translation, and regulation process. (restrictively) - Robert Weaver 分子生物学：从广义上讲就是从分子水平阐明生命现象，狭义上讲就是研究基因的复制、转录，翻译以及调控过程。 What is Molecular Biology?10The relationship of Molecular Biology with other biological SubjectsLife SciencesMedicalSciencesBiologyAgri.Science

8、sMacro(outdoor)Micro(indoor)EcologyEvolutionSystematicsCrop sciencesAnimal husbandryVeterinaryPathology, clinic medicinePreventive medicineetcMolecularBiologyBiochemMicrobiolCellBiologyMolGeneticsDev BiolImmunolMolNeurobiolOthers11分分 子子 生生 物物 学学 发发 展展 历历 史史 （二）（二）现代分子生物学的建立和发展阶段现代分子生物学的建立和发展阶段( (三）三

9、）初步认识生命本质并开始改造生命的深入发初步认识生命本质并开始改造生命的深入发 展阶段展阶段 （一）（一）准备和酝酿阶段准备和酝酿阶段在这一阶段产生了两点对生命本质的认识上的重大突破19世纪后期到20世纪50年代初以1953年Watson和Crick提出的DNA双螺旋结构模型作为现代分子生物学诞生的里程碑50年代初到70年代初70年代后期至今121952年 Franclin 等的X-线衍射分析阐明了核苷酸的空间构像，提出了DNA是螺旋1948-1953年 Chargaff等提出了DNA碱基组成A=T、G=C的Chargaff规则确定了蛋白质是生命的主要物质基础确定了蛋白质是生命的主要物质基础。

10、19世纪末 Eduard Buchner兄第第一次提出酶是生物催化剂。20世纪20-40年代证明酶的本质是蛋白质。陆续发现生命的许多基本现象都与酶和蛋白质相联系1902年 Emil Fisher证明蛋白质结构是多肽；1953年Sanger和Thompson完成了第一个多肽分子胰岛素A链和B链的氨基酸全序列分析。确定了生物遗传的物质是确定了生物遗传的物质是DNADNA1869年 F.Miescher就发现了核素（nuclein）20世纪20-30年代已确认了自然界有DNA和RNA两类核酸，并阐明了核苷酸的组成。1928年Griffith肺炎双球菌感染小家鼠的实验,Avery等证明了肺炎球菌转化因

11、子是DNA。13Eduard BuchnerBerlin, Germany(1860-1917) The Nobel Prize in Chemistry 1907 for his biochemical researches and his discovery of cell-free fermentationHermann Emil Fischer Berlin University Berlin, Germany (1852- 1919)The Nobel Prize in Chemistry 1902 for “in recognition of the extraordinary s

12、ervices he has rendered by his work on sugar and purine syntheses Frederick Sanger, University of Cambridge, United Kingdom(1918-The Nobel Prize in Chemistry 1958 for his work on the structure of proteins, especially that of insulin141869年，德国科学家 Friedrich Meischer是第一个发现DNA的人，他是在病人脓疮中发现这种新化学物质1516 20

13、102010全球十大重要科学成果全球十大重要科学成果2.合成生物学合成生物学: 在生物学和生物技术的一个决定性在生物学和生物技术的一个决定性时刻，研究人员构建了一个合成的基因组，并用时刻，研究人员构建了一个合成的基因组，并用它转变了一种细菌的身份特性。它转变了一种细菌的身份特性。 该基因组取代该基因组取代了该细菌的了该细菌的DNA，使其生产出一组新的蛋白，使其生产出一组新的蛋白质质 这一成就促使国会对合成生物学召开了这一成就促使国会对合成生物学召开了一个听证会。一个听证会。 研究人员预计，将来，定制的合研究人员预计，将来，定制的合成基因组可用来产生生物燃料、医药品或其它有成基因组可用来产生生物

14、燃料、医药品或其它有用的化学制品。用的化学制品。 173.尼安德特人基因组尼安德特人基因组: 研究人员对在研究人员对在3万万8000年至年至4万万4000年前曾经生活在克罗年前曾经生活在克罗地亚的地亚的3个女性尼安德特人的骨头做了尼个女性尼安德特人的骨头做了尼安德特人的基因组测序。安德特人的基因组测序。 对对DNA降解片降解片段进行测序的新方法使得科学家们能够第段进行测序的新方法使得科学家们能够第一次对现代人基因组与我们的尼安德特人一次对现代人基因组与我们的尼安德特人祖先的基因组进行直接的比较。祖先的基因组进行直接的比较。 184.外显子组测序外显子组测序/罕见疾病基因罕见疾病基因: 通过只对

15、通过只对某一基因组中的外显子（或者说是那个极某一基因组中的外显子（或者说是那个极小的实际编码蛋白质的基因组部分）进行小的实际编码蛋白质的基因组部分）进行测序，研究罕见遗传性疾病的研究人员能测序，研究罕见遗传性疾病的研究人员能够发现造成至少够发现造成至少12种疾病的特别的基因突种疾病的特别的基因突变；这些遗传性疾病是由某个单独的有缺变；这些遗传性疾病是由某个单独的有缺陷的基因引起的。陷的基因引起的。 195.黑暗的基因组黑暗的基因组: 基因常常会得到所有的荣基因常常会得到所有的荣耀。耀。 但现在研究人员认识到，这些编码蛋但现在研究人员认识到，这些编码蛋白质的基因区域仅占整个基因组中的白质的基因区

16、域仅占整个基因组中的1.5%。 而其余的基因组部分，其中包括而其余的基因组部分，其中包括小的编码和非编码小的编码和非编码RNA 过去曾被当过去曾被当作作“垃圾垃圾”而勾销而勾销 现在被证明它们现在被证明它们与其它的基因同样地重要。与其它的基因同样地重要。 206.古老的生物性分子古老的生物性分子: 了解像古老的了解像古老的DNA和和胶原质等胶原质等“生物性分子生物性分子” 能够经受好几万能够经受好几万年的时间可为人们提供有关死去已久的植年的时间可为人们提供有关死去已久的植物、动物和人类的重要资讯，这为古生物物、动物和人类的重要资讯，这为古生物学提供了一大裨益。学提供了一大裨益。 对这些极其细小

17、的时对这些极其细小的时光机器所做的分析现在已可披露骨骼上的光机器所做的分析现在已可披露骨骼上的证据所无法提供的诸如某一恐龙羽毛颜色，证据所无法提供的诸如某一恐龙羽毛颜色，或长毛猛犸象如何承受寒冷气温等解剖学或长毛猛犸象如何承受寒冷气温等解剖学上的适应性变化。上的适应性变化。 218.下一世代的基因组学下一世代的基因组学: 更快更廉价的测序更快更廉价的测序技术使人们能够对远古和现代的技术使人们能够对远古和现代的DNA进行进行非常大规模的研究。非常大规模的研究。 例如，例如，1千个基因组千个基因组计划已经发现了令我们人类独一无二的基计划已经发现了令我们人类独一无二的基因组变异因组变异 而其它正在进

18、行中的计划而其它正在进行中的计划一定还会披露更多的基因组功能。一定还会披露更多的基因组功能。 229.微生组微生组: 我们对那些在人体中存活的微生我们对那些在人体中存活的微生物及病毒看法的重大转变使得研究人员产物及病毒看法的重大转变使得研究人员产生了微生组的概念，生了微生组的概念， 也就是说宿主也就是说宿主以及生活在宿主身上或内部的其它生物的以及生活在宿主身上或内部的其它生物的的总体基因组。的总体基因组。 由于我们身体的由于我们身体的90%的的细胞实际上是微生物，科学家们开始了解细胞实际上是微生物，科学家们开始了解微生物基因将会怎样显著地影响我们能从微生物基因将会怎样显著地影响我们能从食物中吸

19、收多少的能量，以及我们的免疫食物中吸收多少的能量，以及我们的免疫系统会如何对感染做出反应。系统会如何对感染做出反应。 23Molecular Biology of the Gene (James D.Watson-)Chapter 20: Techniques of Molecular Biology Chapter 21: Model Organisms24Preparation, analysis and manipulation of nucleic acids25 本章重点：本章重点：1. 1.分子生物学实验主要有哪些？这些实验方法能解决什分子生物学实验主要有哪些？这些实验方法能解决什

20、么问题？么问题？2.2.一些重要实验的原理、过程、关键点。一些重要实验的原理、过程、关键点。3.3.核酸杂交、核酸杂交、DNADNA克隆、克隆、PCRPCR等方法分别要求我们对哪等方法分别要求我们对哪些分子的什么性质有所了解？些分子的什么性质有所了解？4.4.限制性核酸内切酶的来源？性质和特点？应用？限制性核酸内切酶的来源？性质和特点？应用？5.5.分子生物学实验方法中，电泳有几种？各自的特点？分子生物学实验方法中，电泳有几种？各自的特点？6.6.重要的专业英文单词（重要的专业英文单词（200200个以上）个以上）26The living cell is an extraordinarily

21、complicated entity.Understanding how the genetic processes of the cell work requires powerful,and complementary experimental approaches including the use of suitable model organisms in which the tools of genetic analysis are available.27nThe methods of molecular biology depend upon understanding of

22、the properties of biological macromolecules themselves.nUsing computational or bioinformatics approaches,to undertake large-scale genomic comparisons of both the coding and noncoding regions of various organisms.28The methods depend upon the understanding of the properties of biological macromolecul

23、es themselves.Hybridization-the base-pairing characteristics of DNA and RNADNA cloning- DNA polymerase, restriction endonucleases and DNA ligasePCR-Thermophilic DNA polymerase291.Electrophoresis2.Restriction3.Hybridization4.PCR 5.DNA Cloning and gene expression6.Genome sequence & analysis 30Gel

24、matrix is an inerted, jello-like porous material that support and allows macromolecules to move through. Agarose and polyacrylamide are two different gel matricesElectrophoresisElectrophoresis31DNA and RNA molecules are negatively charged, thus move in the gel matrix toward the positive pole (+)Line

25、ar DNA molecules are separated according to sizeThe mobility of circular DNA molecules is affected by their topological structures. The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled linear nicked or relaxed Electrophoresis32Fig 20-1: DNA separation by gel e

26、lectrophoresislargemoderate smallEthidium bromide33schematic of electrophoretic separation of DNA topoisomers34To separate DNA of different size rangesnNarrow size range of DNA: use polyacrylamidenWide size range of DNA: use agarose gelnVery large DNA(30-50kb): use pulsed-field gel electrophoresisEl

27、ectrophoresis35pulsed-field gel electrophoresisSwitching between two orientations: the larger the DNA is, the longer it takes to reorientElectrophoresisElectrophoresis36Two alternative kinds of gel matrices: Polyacrylamide(聚丙烯酰胺聚丙烯酰胺):has high resolving capability but can separate DNAs only over a n

28、arrow size range. Can resolve DNAs that differ from each other in size by as little as a single base pair but only with molecules of up to several hundred base pairs.Agarose(琼脂糖琼脂糖)has less resolving power than polyacrylamide but can separate from one another DNA molecules of up to tens,and even hun

29、dreds,of kilobases.37Electrophoresis separates DNA molecules according to their molecular weight,shape topological properties. It is used to separate RNAs.RNAs have a uniform negative charge,but RNA usually single-stranded and have extensive secondary and tertiary structure.to deal with this ,RNAs c

30、an be treated with reagents(glyoxal,乙二醛乙二醛) to prenvent the formation of base pairs.38Why use endonucleases?-To make large DNA molecules break into manageable fragmentsRestriction digestionRestriction digestion39nRestriction endonucleases: the nucleases that cleave DNA at particular sites by the rec

31、ognition of specific sequencesnThe target site recognized by endonucleases is usually palindromic (回回文结构文结构). e.g. EcoRI5.GAATTC.3 .CTTAAG.Restriction digestion40Restriction endonuclease(Restriction enzyme)1.Bacterial enzymes which cut DNA into defined and reproducible fragments2.Identified in the l

32、ate 1960s3.Key discovery which allowed the DNA cloning to become a reality41One component of the bacterial restriction-modification system, a natural defense mechanism of bacteria to against the introduction of foreign DNA into the cellRestriction endonuclease: recognize a short, symmetrical DNA seq

33、uence, and cut DNA backbone in each strand at a specific site within that sequence (kill foreign DNA)Mythylase: methylates C or A of the cellular DNA Restriction endonuclease(origination)42E. coli K strain: restricted replication of lambdaE. coli C strain: efficient replication of lambdaRestriction

34、enzyme (Lederberg & Meselson, 1964) First restriction enzyme from E. coli K (Meselson & Yuan, 1968) First type II restriction enzyme from Haemophilus influenzae (Smith, 1970)43Types of Restriction endonucleaseType IType IIType IIIFunctionsEndonuclease & methylaseEndonucleaseEndonucleaseC

35、onditionsATP, Mb2+Mg2+ATP, Mg2+Recognition sequencesEcoK: AACN6GTGCEcoB: TGAN8TGCTpalindromicEcoP1: AGACCEcoP15: CAGCAGCutting sitesAt least 1000bp awayAt or close to recog. seq24-26 bp away44 Restriction endonucleases (TypeII REs 2500 isolated,more than 300 available in lab )45nTo name a restrictio

36、n endonuclease: e.g. EcoRIthe 1st such enzyme foundEscherichia coli Species categoryR13strainRestriction digestionRestriction digestion46nFrequency of the occurrence of hexamaeric sequence: 1/4096 (4-6) Randomly Restriction digestionRestriction digestion47nConsider a linear DNA molecule with 6 copie

37、s of GAATTC: it will be cut into 7 fragments which could be separated in the gel electrophoresis by size(The largest fragment)(The smallest fragment)Fig 20-3 digestionof a DNA fragment with endonuclease EcoRI48Restriction digestion49nEndonucleases are used to make restriction map:ne.g. the combinati

38、on of EcoRI + HindIIInAllows different regions of one molecule to be isolate and a given molecule to be identifiednA given molecule will generate a characteristic series of patterns when digested with a set of different enzymes Restriction digestionRestriction digestion505152Different enzymes recogn

39、ize their specific target sites with different frequencynEcoRI Recognize hexameric sequence: 4-6nSau3A1 Recognize terameric sequence: 4-4nThus Sau3A1 cuts the same DNA molecule more frequentlyRestriction digestionRestriction digestion53Some Restriction Endonucleases and Their Recognition Sequences E

40、nzyme Sequence Cut frequency Sau3A1 5-GATC-3 0.25kb EcoR1 5-GATTC-3 4kb Not1 5-GCGGCCGC-3 65kb54sticky ends blunt ends Fig 20-4 recognition sequences and cut sites of various endonucleasesRestriction digestionRestriction digestion55nThe 5 protruding ends of are said to be “sticky” because they readi

41、ly anneal through base-pairing to DNA molecules cut with the same enzymeReanneal with its complementary strand or other strands with the same cut Restriction digestionRestriction digestion56 Hybridization: the process of base-pairing between complementary ssDNA or RNA from two different sourcesDNA h

42、ybridizationDNA hybridization57Hybridization Can Be Used to Identify a Specific Clone in a DNA LibraryUsing a DNA probe whose sequence matches part of the gene of interest.such a probe can be used to identify colonies of cells harboring clones containing that region of the gene.Colony hybridization

43、a labeled DNA probe is used to screen a library.58Probe(探针)be used to search mixtures of nucleic acids for molecules containing a complementary sequence. The probe DNA must be labeled so that it can be readily located once it had found its target sequence.Labeling of DNA or RNA probes End labeling:

44、put the labels at the endsUniform labeling: put the labels internally radioactive labeling: display and/or magnify the signals by radioactivity Non-radioactive labeling: display and/or magnify the signals by antigen labeling antibody binding enzyme binding - substrate application (signal release)End

45、 labelingSingle stranded DNA/RNA5-end labeling: polynucleotide kinase (PNK)3-end labeling: terminal transferaseLabeling at both ends by kinase, then remove one end by restriction digestion -G-CTTAAp55pAATTC GSouthern and Northern blottingDNA on blotRNA on blot1. Genomic DNA preparation RNA preparati

46、on2.Restriction digestion -3.Denature with alkali - 4. Agarose gel electrophoresis 5. DNA blotting/transfer and fixation RNA6. Probe labeling 6. Hybridization (temperature) 7. Signal detection (X-ray film or-) Southern analysis65Northern blot hybridization be used to identify a particular mRNA in a

47、population of RNAs.Use northern blot hybridization to ask how much more mRNA of a specific type is present in a cell treated with an inducer of the gene in question compared to an uninduced cell.Blot type TargetProbeApplicationsSouthern DNA DNA or RNAmapping genomic clonesestimating gene numbersNort

48、hernRNADNA or RNARNA sizes, abundance,and expressionWesternProteinAntibodiesprotein size, abundance67 4.Polymerase chain reaction: （聚合酶链反应）（聚合酶链反应） Amplifies DNAs by Repeated Rounds of DNA Replication in Vitrois a powerful method for amplifying particular segments of DNA. 68nPCRnThe PCR cycle （循环）（循

49、环）nTemplate （模板）（模板）nPrimers （引物）（引物）nEnzymes （酶）（酶）nPCR optimization （优化）（优化）69PCRThe polymerase chain reaction ( (聚合酶链式反应聚合酶链式反应) ) (PCR)：to amplify a sequence of DNA using a pair of primers each complementary to one end of the the DNA target sequence.70The PCR cyclenDenaturation （变性）（变性）: The tar

50、get DNA (template) is separated into two strands by heating to 95nPrimer annealing （引物退火）（引物退火）: The temperature is reduced to around 55 to allow the primers to anneal.nPolymerization (elongation, extension): The temperature is increased to 72 for optimal polymerization step which uses up dNTPs and required Mg+.7172Primers 引物引物n PCR primers：about 18 to 30 nt long and with similar G+C contents.n Tm=2(a+t)+4(g+c): determine annealing temperature. If the primer is 18-30 nt, annealing temperature can be Tm 5oC7374Enzymes for PCRnThe most common is Taq polyme