《分子生物学》2sectionggenemanipulation1精选课件

1、DNA Cloning DNA cloning: DNA克隆Molecular cloning： 分子克隆Gene manipulation:基因操作Recombinant DNA technology：重组DNA技术Genetic engineering遗传工程，基因工程Biotechnology：生物技术(Gene engineering, Enzyme engineering, Cell / Fermentation engineering, Biomedicine, Agrobiotechnolgy etc.)第1页，共55页。DNA cloning (Gene manipulatio

2、n)To place a relatively short fragment of a genome, which might contain the gene or other sequence of interest, in an autonomously replicating piece of DNA, known as a vector （载体）, forming recombinant DNA, which can be replicated independently of the original genome, and normally in other host speci

3、es altogether. Propagation of the host organism containing the recombinant DNA forms a set of genetically identical organism, or a clone. This process is called DNA cloning.第2页，共55页。Basic procedureof DNA cloningVector第3页，共55页。Genomic fragment (restriction, PCR), cDNA (insert)Plasmid preparation (vec

4、tor)Restriction digestion (trimming the DNA ends) Ligation (join the insert and the vector) Transformation (introduce the plasmids into host cells)Analysis of the recombinants Electrophoresis (check your DNA)DNA Cloning: a simplified flow chart 第4页，共55页。Isolation and manipulation of fragments of an

5、organisms genomeMolecular analysis of proteins or other interested gene products Impossible by direct purification DNA cloning Impossible by direct isolationCrucial ! Make all possible ! Gene manipulation, molecular cloning, genetic engineering第5页，共55页。Applications of DNA cloningSequencing, hence to

6、 derive protein sequence; genomicsIsolation and analysis of gene promoter etcInvestigation of protein/enzyme/RNA function in various forms.Identification mutations, genetic diseasesBiotechnology: proteins of pharmaceutical importanceTransgenic plants and animalsGene therapy第6页，共55页。DNA Cloning, Tech

7、niques & ApplicationsSection G: Gene manipulation (DNA cloning & subcloning, & basic techniques)section H: Cloning vectorsSection I: Gene libraries & screening Section J: Analysis & uses of cloned DNA The most basic concepts and technical tools of molecular biology 第7页，共55页。G1 DNA cloning: an overvi

8、ew (basic concepts)G2 Preparation of plasmid DNAG3Ligation, transformation and analysis of recombinants Section G Gene manipulation第8页，共55页。G1 DNA cloning: an overviewHosts and vectorsSubcloningDNA librariesScreening librariesAnalysis of a cloneback第9页，共55页。Plasmid as vectorsPlasmids （质粒）: small, ex

9、trachromosomal circular molecules, from 2 to 200 kb in size, which exist in multiple copies within the host cells.contain an origin of replication and replicate independentlyUsually carry a few genes, one of which may confer resistance to antibacterial substance.Example: ampR gene encoding the enzym

10、e b-lactamase which degrades penicillin antibiotics such as ampicillin; kanR for kanamycin.第10页，共55页。backEarlier plasmid developed第11页，共55页。Versatile cloning plasmidPhagemid(噬菌粒）第12页，共55页。第13页，共55页。第14页，共55页。Hosts and vectorsHost organism/cell: where the plasmids get multiplied and propagated faithf

11、ully, which is crucial for DNA cloning.Hosts for DNA cloning vectorProkaryotic host : E. coli ( most cases)Eukaryotic host : Yeast Saccharomyces cerevisiae (large fragments of human genome)第15页，共55页。General features of a Vectorautonomously replicating DNA independent of hosts genome.Easily to be iso

12、lated from the host cellMost are circular, some are linearContains at least one selective marker, which allows host cells containing the vector to be selected amongst those which do not. Contains a multiple cloning site (MCS)第16页，共55页。Types of vectorsCloning vectorsExpression vectorsIntegration vect

13、orsViral vectors第17页，共55页。Cloning vectors: allowing the exogenous DNA to be inserted, stored, and manipulated at DNA level. E. coli cloning vector: plasmids, bacteriophages (l and M13), plasmid-bacteriophage l hybrids (cosmids).Yeast cloning vector: yeast artificial chromosomes (YACs)第18页，共55页。MCSEx

14、pression vectors: allowing the exogenous DNA to be inserted and expressed. Promoter and terminator for RNA transcription are required. bacterial expression vectors yeast expression vectors mammalian expression vectors第19页，共55页。第20页，共55页。Integration vectors: allowing the exogenous DNA to be inserted

15、and integrated into a chromosomal DNA after a transformation. The integration is either random insertion or conducted by homologous recombination between the homologous sequence shared by the plasmid and the genome of the recipient cells. bacterial integration vectors (Agrobacterium tumefaciens Ti p

16、lasmid is used to integrate DNA into plant genome) yeast integration vectors Mammalian integration vector: gene targeting back第21页，共55页。Viral vectors:. Bacterial phage: Lambda, M13 Insect: baculoviruses Mammalian viruses: SV40, pox virus, adenovirus, retroviruses Plant viruses: TMV, PVXback第22页，共55页

17、。Adenoviral vector system第23页，共55页。Plant virus vector: PVX (potato virus X)第24页，共55页。Subcloning Transfer of a fragment of cloned DNA from one vector to another.Enables us to investigate a short region of a large cloned fragment in more detail.To transfer a gene from one plasmid to a vector designed

18、to express it in a particular species. 第25页，共55页。Preparation of plasmids containing a cloned DNA fragment (insert)Plasmid preparation (vector)Restriction digestion (trimming the DNA ends) Separation, purification, ligation (join the insert and the vector) Transformation & selection of transformants(

19、introduce the plasmids into host cells)Analysis of the recombinants DNA Subcloning: a flow chart Restriction endonuclease第26页，共55页。backAgrose Gel Electrophoresis: check your DNA at each stepSeparation and Purification of DNA fragments of interestsAnalysis of recombinant plasmidsladderRestriction ana

20、lysis of a plasmid第27页，共55页。DNA libraries DNA libraries are sets of DNA clones, each of which has been derived from the insertion of a different fragment into a vector followed by propagation in the host. A clone is a genetically distinct individual or set of identical individualsGenomic librariescD

21、NA libraries第28页，共55页。Genomic librariesprepared form random fragments of genomic DNA, which may be inefficient to find a gene because of the huge abundance of the non-coding DNAcDNA libraries DNA copies (cDNA) synthesized from the mRNA by reverse transcription are inserted into a vector to form a cD

22、NA library. Much more efficient in identifying a gene, but do not contain DNA coding for functional RNA or noncoding sequence.第29页，共55页。Screening libraries Colony or plaque hybridization:Radiolabeled probes complementary to a region of the interested geneProbes: An oligonucleotide derived from the s

23、equence of a protein product of the geneA DNA fragment/oligo from a related gene of another speciesPCR productPlating the cells carrying the library Colony or plaque lift on membrane and then hybridize with the labeled probeSearching the genes of interest in a DNA library 第30页，共55页。Screening librari

24、es Expression screening:Specific antibody to gene product Screening library by PCR.:Specially prepared libraryFunctional screening.:Complementation to a lethal phenotype, possible for other kinds of positive screeningSearching the genes of interest in a DNA library 第31页，共55页。Identify the protein pro

25、duct of an interested geneProtein activityWestern blotting using a specific antibodyIn vivo expression and functional assayback第32页，共55页。Analysis of a clone Restriction mapping: digestion of the with restriction enzymes.Sequencing the cloned DNAbackYou may have to fully understand the function and a

26、pplication of all the enzymes listed in Table 1 if you want to manipulate genes第33页，共55页。Enzymes commonly used in DNA cloning Alkaline phosphotaseReverse transcriptase, DNA ligase (T4)DNA pol I (Klenow fragment), T4, TaqExunuclease III Mung bean nuclease and S1 nucleasePolynucleotide kinaseRestricti

27、on enzymes: e.g. EcoRI, HindIIIRNase A, RNase HT7, T3and SP6 RNA polymerases Terminal transferase第34页，共55页。backG2. Preparation of plasmid DNA 1.Plasmid as vectorsPlasmid minipreparationAlkaline lysisPhenol extractionEthanol precipitationCesium chloride gradient purification第35页，共55页。Plasmid as vecto

28、rsPlasmids: small, extrachromosomal circular molecules, from 2 to 200 kb in size, which exist in multiple copies within the host cells. contain an origin of replication and replicate independentlyUsually carry a few genes, one of which may confer resistance to antibacterial substance.Example: ampr g

29、ene encoding the enzyme b-lactamse which degrades penicillin antibiotics such as ampicillin.第36页，共55页。Plasmid minipreparation from E. coliPlasmids2-20 kb in length that is much smaller than E. coli chromosomal DNA (4600 kb), and independently supercoiledResistant to shearing force and chemical denat

30、uration, thus can be isolated from the chromosomal DNA easily such as by alkaline lysis.Minipreparation (miniprep)Isolation of plasmid DNA from a few mililiters (ml) of bacterial culture. 第37页，共55页。MiniprepsGrowth of the cells containing plasmidsCollect the cells by centrifugationAlkaline lysis resu

31、spension alkaline lysis neutralizationPhenol extraction to get rid of the protein contaminantsEthanol precipitation to concentrate the nucleic acids remained (0.3M NaAc, 2-3 vol ethanol). Resuspend in suitable buffer: TE10/1, pH 8.0 (Please note that RNase A is very bad for the lab working with RNA)

32、 第38页，共55页。Alkaline lysis Resuspend the cells in a buffer solutionLysozyme to digest the cell wall (optional)Cell lysis in lysis buffer containing SDS (disrupts cell membrane and denatures proteins) and NaOH (denatures DNA)Neutralization buffer containing KOAc (pH 5): renaturation of plasmid DNA (su

33、percoiled) and precipitation of denatured proteins and chromosomal DNA.Centrifugation plasmid in supernatant (lysate)第39页，共55页。Grow the cellHarvest the cell by centrifugationAlkaline lysis of the cellResuspend the cell pelletneutralizationPhenol extractionEthanol precipitationCsCl gradient purificat

34、ion第40页，共55页。Purification of plasmid DNA by Cesium chloride gradient centrifugationCsCl gradient purification is the last step of large scale plasmid DNA purificationLaboriousBest for the production of very pure supercoiled plasmid DNAThe presence of ethidium bromide (EB) is important. Binding of EB

35、 to DNA will unwind the DNA and reduce the DNA densitySupercoilded DNA bind less EB than linear DNA or nicked DNA, thus has a higher densitySupercoiled DNA may be purified from protein, RNA chromosomal DNA and nicked plasmid DNA in one step!back第41页，共55页。Agarose gel electrophoresissupercoilednicked第

36、42页，共55页。Isolation of fragments and Agarose gel electrophoresisinsertRestriction digestionAgarose gel electrophoresis3.Gel excision and purificationLigation with vectorTransformationback第43页，共55页。G4. Ligation, transformation and analysis of recombinantsAlkaline phosphataseDNA ligation & recombinant

37、DNA moleculesTransformation & selection Transformation efficiency4.Screening transformants5.Growth and storage of transformantsGel analysisFragment orientation第44页，共55页。Alkaline phosphataseSingle restriction enzyme directed cloning Removes the phosphate groups from the 5-ends of the vector DNA linea

38、rized by a single restriction enzyme to prevent the self-ligation of the vector DNA upon the followed ligation第45页，共55页。DNA ligation Covalently join the DNA molecules with the base-pairing cohesive ends, or blunt ends, if the 5-ends have phosphate groups. 第46页，共55页。 X if the vector is phosphorylated

39、Recombinant DNA molecules第47页，共55页。The use of alkaline phosphatase to prevent religation of vector moleculesG -OHCTTAA -OH第48页，共55页。Transformation and selectionCompetent cells (感受态细胞): E. coli cells treated with Ca2+ solution are susceptible to take up exogenous DNA. Enzymes involved in host cell defending, such as restriction-modification system are suppressed. Transformation （转化）: a process of uptake of exogenous DNA by competent cells. Heat-shock （热休克）: After the DNA is uptaken, the cells shall be put at 42C for 1-2 min