[自然科学]Technique of molecular biology.ppt

Molecular Biology of the Gene, 6/E - Watson et al. (2007) Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods 1 The revised central dogma Translation RNA processing DNA repair and recombination 基 因 组 的 保 持 基 因 组 的 表 达 2 Chapter 20Chapter 20 Techniques of Techniques of Molecular Biology Molecular Biology 3 Techniques of Molecular BiologyTechniques of Molecular Biology nseparating individual macromoleculars from the myriad mixtures in cell ndissecting the genome into manageable sized segments for manipulation and analysis of specific DNA sequences n n Techniques of molecular biology have Techniques of molecular biology have been one of the major driving forces in been one of the major driving forces in the research of molecular biologythe research of molecular biology 4 The methods depend upon understanding of the properties of biological macromolecules themselves. Hybridization(分子杂交）-the base- pairing characteristics of DNA and RNA DNA cloning- DNA polymerase, restriction endonucleases and DNA Ligase PCR-Thermophilic DNA polymerase 5 Outline nTopic 1: Nucleic acids TechniquesNucleic acids Techniques nTopic 2: Protein TechniquesProtein Techniques nTopic 3: Techniques for studying Techniques for studying DNA-protein interactionprotein interaction nTopic 4: Techniques for studying Techniques for studying proteinprotein-protein interactionprotein interaction Basic principleBasic procedure application 6 Topic 1: Nucleic acids TechniquesNucleic acids Techniques 1.Separation by Electrophoresis (电泳分离) 2.Cut by Restriction endonuclease (限制性 内切酶切割) 3.Identification by Hybridization (杂交鉴 定) 4.DNA Cloning and gene expression (基因 克隆和表达技术） 5.PCR（聚合酶链式反应） 7 Gel electrophoresis separates DNA and Gel electrophoresis separates DNA and RNA molecules according to RNA molecules according to sizesize, , shapeshape and and topological propertiestopological properties 1. Gel Electrophoresis （凝胶电泳） 8 1.DNA and RNA molecules are negatively charged, thus move in the gel matrix ( 胶支持物) toward the positive pole (正电 极) 2.Linear DNA molecules2.Linear DNA molecules are separated according to sizessizes. The large DNA molecules move slower than the small molecules. DNA gel mobility (electrophoretic mobility ， DNA在胶上的迁移率) 9 largemoderate small Run gel(跑胶) 10 3.The mobility of circular DNA moleculescircular DNA molecules is affected by their topological structures. The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled (超螺旋) linear (线性) nicked or relaxed (缺刻或松散) 11 DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium bromide (EB,溴化乙锭) DNA is separated by gel electrophoresis 1 kb 0.5 kb 2 kb 3 kb 4 kb DNA marker 12 Gel matrix (胶支持物) is a jello(果冻状）- like porous material that supports and allows macromolecules to move through. Gel matrix (胶支持物) Polyacrylamide (聚丙烯酰胺) Agarose (琼脂糖) 13 Polyacrylamide (聚丙烯酰胺 ): (1)has high resolving capability(分辨力高), and can resolve DNA/RNA that differ from each other as little as a single base pair. (2)but can separate DNA over a narrow size range (up to a few hundred bp, 几百bp). 14 Agarose (琼脂糖): (1)a much less resolving power than polyacrylamide (2)but can separate DNA molecules of up to tens of kb 1 kb 0.5 kb 2 kb 3 kb 4 kb 15 (1)RNA have a uniform negative charge as DNA does. (2)RNA is single-stranded and have extensive secondary and tertiary structure, which significantly influences their electrophoretic mobility. (3)RNA can be treated with reagent such as glyoxal (乙二醛) to prevent RNA base pairing, so that its mobility only correlates with the molecular weight Electrophoresis is also used to separate RNAs 16 2. Restriction endonucleases 2. Restriction endonucleases ( (限制性内切酶限制性内切酶) ) cleave DNA cleave DNA molecules at particular sitesmolecules at particular sites nWhy use endonucleases? nTo make large DNA molecules break into manageable fragments nWhy use restriction endonucleases? ncleave DNA molecules at particular sites 17 n n Restriction endonucleases (RE) Restriction endonucleases (RE) are the nucleases that cleavecleave DNA at particular sites by the recognitionrecognition of specific sequences.(剪刀) nRE used in molecular biology typically recognize (识别) short (4-8bp) target sequences that are usually palindromic (回文结构), and cut (切割) at a defined sequence within those sequences. 18 5.GAATTC.3 .CTTAAG. EcoRI 35 反向重复序列（回文结构） 19 the 1st such enzyme found Escherichia coli Species category R13 strain How to name a restriction endonuclease? EcoRI 20 na linear DNA molecule with 6 copies of GAATTC nit will be cut into 7 fragments which could be separated by the gel electrophoresis. (The largest fragment)(The smallest fragment) Digestionof a DNA fragment with endonuclease EcoRI 21 nRestriction enzymes differ in the recognition specificityrecognition specificity: target sites are different. (2) Restriction enzymes differ in the length the length they recognizedthey recognized, and thus the frequencies differ: Sau3AI, 5-GATC-3; NotI, 5- GCGGCCGC-3 (3) Restriction enzymes differ in the nature the nature of the DNA ends they generateof the DNA ends they generate: blunt/flush ends (平末端), sticky/staggered ends (粘性末端). 22 sticky ends (粘性末端) blunt ends (平末端) Recognition sequences and cut sites of various endonucleases 23 Cleavage of an EcoRI site: The 5 protruding ends are said to be “sticky” because they readily anneal (退火) through base-pairing to DNA molecules cut with the same enzyme Gene B Gene A 24 25 Discovery of restriction endonucleases Research strategy: from phenomenon to mechanism Phenomenon Hypothesis Experimental proof Mechanism Practical use of restriction endonucleases 26 Salvador E. Luria, 1969 Nobel prize in Physiology or Medicine Model: Phage infecting E.coli Phenomenon: restriction and modification in bacteria (限制与修饰现象） 27 Werner Arber, 1978 Nobel Prize in Physiology or Medicine Discovery of restriction enzymes, which are the tools which make it possible to open the sealed book He analyzed an apparently obscure phenomenon in bacteria, called host-controlled modification and restriction Arber postulated that bacteria contain restriction enzymes with the capacity to recognize and degrade foreign DNA- phage DNA (protect the host from foreign genes) 28 Arbers Hypothesis nBacteria have the ability to restrict phage (virus) infection by cutting up the phage DNA once its injected into the cell nThe enzyme that cuts phage DNA is called a restriction enzyme, or more properly, a restriction endonuclease 29 Hamilton O. Smith, 1978 Nobel Prize in Physiology or Medicine Hamilton Smith verified Arbers hypothesis- bacteria contain restriction enzymes with the capacity to recognize and degrade foreign DNA He purified one restriction enzyme and showed that it could cleave foreign DNA He determined the chemical structure of the regions of DNA which were cut by the restriction enzyme (The enzymes dont cut randomly. They bind to specific sequences and only cut at those sites-识别回文结构) 30 nSmith, H.O. and Wilcox, K.W. A restriction enzyme from Hemophilus influenzae. 1. Purification and general properties. J. Mol. Biol. 51, 379 (1970). nKelly, T.J., Jr. and Smith, H.O. A restriction enzyme from Hemophilus influenzae. 11. Base sequence of the recognition site. J. Mol. Biol. 51, 393 (1970). nRoy, P.H. and Smith, H.O. The DNA methylases of Hemophilus influenzae Rd. 1. Purification and properties. J. Mol. Biol. 81, 427 (1973). nRoy, P.H. and Smith, H.O. The DNA methylases of Hemophilus influenzae Rd. 11. Partial recognition site base sequences. J. Mol. Biol. 81, 445 (1973). nPapers 31 Daniel Nathans, 1978 Nobel Prize in Physiology or Medicine The last step in this development was taken by Dan Nathans He pioneered the application of restriction enzymes in genetics and his work has been a source of inspiration for scientists who subsequently started to use restriction enzymes He constructed the first genetic map using restriction enzymes by cleaving the DNA from a monkey virus-SV40 32 nAs I look back on the last few decades of my life, I am struck by the good fortune that came my way. nThroughout my schooling there was an abundance of opportunity and encouragement. nSeveral of my teachers were remarkable individuals who had a lasting influence on me. nAt every stage of my career I have had interesting and cordial colleagues, some of whom are close friends. nMy field of research is as exciting to me as ever, and it remains essentially a “cottage industry“ effort. nI have had talented students who are a source of much enjoyment, and I anticipate more to come as their careers develop. nAnd most important, my wife and sons have created in our home an atmosphere of joy and harmony, so essential to everything else. 33 34 nArber discovered restriction enzymes. He postulated that these enzymes bind to DNA at specific sites containing recurring structural elements made up of specific basepair sequences. n Smith verified Arbers hypothesis with a purified bacterial restriction enzyme and showed that this enzyme cuts DNA in the middle of a specific symmetrical sequence. Other restriction enzymes have similar properties, but different enzymes recognize different sequences. n Nathans pioneered the application of restriction enzymes to genetics. He demonstrated their use for the construction of genetic maps and developed and applied new methodology involving restriction enzymes to solve various problems in genetics. 35 nThis years Nobel Prize in medicine or physiology is awarded for discoveries with far reaching consequences for genetics. The task of genetics is to describe and explain how genes are organized and expressed in cells and in living organisms. The discovery of restriction enzymes provided new tools for the detailed chemical analysis of the mechanism of gene action. Even though these enzymes have been available only during a few years their application to genetics has already led to new and far reaching results, in particular concerning the organisation and expression of genes (= DNA) of higher animals. All work in this area carried out by many research groups all over the world, is based on the discoveries made by the three laureates. Restriction enzymes are used as tools to dissect DNA into smaller defined fragments. These can be used to determine the order of genes on chromosomes, to analyse the chemical structure of genes and to recombine genes by chemical means. Most important restriction enzymes are used to analyse the function of regions of DNA which regulate gene expression. This opens up new areas of research to study the connection between heredity and function. We can now begin to answer questions of central biological importance in developmental biology: how do genes direct the evolution of a single fertilized egg to a complete individual with many different organs? What determines that the cells within one organ normally retain their specialized functions? Different diseases are expressions of disturbances in normal functions and increased knowledge in molecular genetics should aid in preventing and treating malformations, hereditary diseases and cancer. Werner Arber started this field of research in Geneva during the 1960s. He discovered restriction enzymes. Arber was studying an earlier known phenomenon, “host controlled restriction of bacteriophages“, and found that this process involved changes in the DNA of the virus. The process apparently served to form a barrier against foreign genetic material. Arber showed that the phenomenon could be divided into two components: restriction and modification. Restriction involved a breakdown of DNA, modification was a change (= methylation) of DNA which prevented restriction. Arber postulated that both processes are catalyzed by specific restriction and modification enzymes. He proposed that DNA molecules contain specific sites with the capacity to bind both types of enzymes. These sites are created by recurring structural elements formed from specific basepair sequences. The enzymes act at these sites either by cleaving the molecule (= restriction) or by methylating it (= modification). Hamilton Smith verified Arbers hypothesis. He is a biochemist and worked independently of Arber in Baltimore. In 1970 he published two classical papers which described the discovery of a restriction enzyme from the bacterium Heamophilus influenzae and characterized in detail the mechanism of its action. Other scientists before Smith had unsuccessfully tried similar experiments. The restriction enzyme from Haemophilus influenzae degrades foreign DNA to large fragments, about 1000 basepairs in size, but does not touch the DNA of the host bacterium. Most important, Smith showed that all fragments at their beginning and end had the same three basepairs showing that the enzyme had cleaved DNA wherever a specific sequence of 6 basepairs was present. This sequence was internally symmetric and was cleaved in the middle. Many other restriction enzymes have by now been characterized by others using the methodology worked out by Smith. More than 100 such enzymes are known and in most cases the same pattern is observed: a restriction enzyme recognizes certain symmetrical basepair sequences and cleaves DNA wherever these sequences occur. Different enzymes recognize different sequences and by now a battery of enzymes is available which can be used to cleave DNA at different sites in order to produce a multitude of defined fragments. Dan Nathans pioneered the application of restriction enzymes to problems of genetics. He works in Baltimore at the same university as Smith. All his contributions in this area of research were made during the 1970s. Nathans uses in his experiments the small DNA from a simian virus, called SV40, but his results are of general significance. In his first communication from 1971 he showed that the restriction enzyme discovered by Smith cleaves SV40 DNA into 11 well defined fragments. In this communication Nathans also discussed other possible applications of restriction enzymes in genetics and in a brilliant way predicted much of the later development. Nathans publication from 1971 no doubt served as a major source of inspiration for scientists who subsequently started to use restriction enzymes. Two years later he described the cleavage patterns of SV40 DNA obtained with two additional restriction enzymes. He could then piece together the fragments obtained from the three cleavages and construct the complete genetic map of SV40 DNA, the first obtained by a chemical method. The general approach designed by Nathans for SV40 was later used by other scientists for mapping increasingly complex DNA structures. The map of SV40 DNA was further refined by other scientists. Today we know the complete nucleotide sequence of the molecule and thus can write the complete chemical formula for all the genes of an animal virus. Nathans himself continuously contributed new ideas and developed new methods for the application of restriction enzymes to genetic problems and has continuously been a main source of inspiration in this field of research. 36 Discovery of restriction endonucleases Phenomenon Hypothesis Experimental proof Mechanism practical use of restriction endonucleases 37 How does the bacteria prevent its own DNA from being cut by the restriction endonucleases? nModification nOne of the nucleotides in bacteria DNA is modified by an methylase enzyme (甲基化酶，modification enzyme) that attaches a methyl group to one of the bases nThe restriction enzyme doesnt bind to sites where one of the bases is modified by methylation 38 Restriction Modification 39 3. DNA hybridization can be 3. DNA hybridization can be used to identify specific DNA used to identify specific DNA moleculesmolecules Hybridization (杂交）: the process of base-pairing between complementary ssDNA or RNA from two different sources. 40 A labeled, defined sequence used to search mixtures of nucleic acids for specific molecules containing a specific sequence which is complementary to probe Probe (探针) 5-GAATTTGCGTATGCG-3 3-CTTAAACGCATACGC-5Probe 41 n n DNA hybridization using probes can DNA hybridization using probes can be used to be used to identify specific sequenceidentify specific sequence in the complicated mixture of nucleic in the complicated mixture of nucleic acidsacids 42 Radioactive labeling: display and/or magnify the signals by radioactivityradioactivity. eg. 32P Non-radioactive labeling: display and/or magnify the signals by antigen labelingantigen labeling antibody binding enzyme binding - signal detection eg. DIG(地高辛标记)-抗体抗原特异性反应 Probe (探针) must be labelled (why labeling?) 43 Southern hybridization nBasic principle: the base- pairing characteristics of DNA and DNA nApplication: identify specific identify specific sequence in the complicated sequence in the complicated mixture of DNA, mixture of DNA, estimating gene numbers 44 DNA 样品 DNA 探针 变性 X-ray 片 限制性内切 酶消化 琼脂糖凝胶电泳 转移印迹 胶 膜 标记 杂交 曝光 blot 45 46 Example: 实战中学习技术 nProblem: identify whether the genome of one fungi contains specific gene sequence coding laccase（漆酶） nHow to use Southern hybridization hybridization to resolve this problem?to resolve this problem? 47 nProbe: DNA sequence specific to the known laccase gene Labeled probe（标记探针） 48 Southern hybridization 49 Northern hybridization nBasic principle: the base-pairing characteristics of DNA and RNA nApplication: Identify specific sequence in the Identify specific sequence in the complicated mixture of complicated mixture of RNARNA Detecting the expression level of specific gene mRNA contents (mRNA 丰度) expression level of specific gene 50 Example: 实战中学习技术 nProblem: measuring the expression level of laccase gene in different stages of fungal development nHow to use Northern hybridization hybridization