《分子生物学》2-section g gene manipula

1、编辑pptDNA Cloning DNA cloning: DNA克隆克隆Molecular cloning： 分子克隆分子克隆Gene manipulation:基因操作基因操作Recombinant DNA technology：重组重组DNA技术技术Genetic engineering遗传工程，基因工程遗传工程，基因工程Biotechnology：生物技术生物技术(Gene engineering, Enzyme engineering, Cell / Fermentation engineering, Biomedicine, Agrobiotechnolgy etc.)编辑ppt

2、DNA cloning (Gene manipulation)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）（载体）, forming recombinant DNA, which can be replicated independently of the original genome,

3、 and normally in other host species 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.编辑pptBasic procedureof DNA cloningVector编辑pptGenomic fragment (restriction, PCR), cDNA (insert

4、)Plasmid preparation (vector)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 编辑pptIsolation and manipulatio

5、n of fragments of an organisms genomeMolecular analysis of proteins or other interested gene products Impossible by direct purification DNA cloning Impossible by direct isolationCrucial ! Crucial ! Make all possible ! Make all possible ! Gene manipulation, molecular cloning, genetic engineering编辑ppt

6、Applications of DNA cloningSequencing, hence to 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 an

7、d animalsGene therapy编辑pptDNA Cloning, Techniques & 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 technica

8、l tools of molecular biology 编辑pptG1 DNA cloning: an overview (basic concepts)G2 Preparation of plasmid DNAG3Ligation, transformation and analysis of recombinants Sectctio on G Gene ma manipulatpulatio on编辑pptG1 DNA cloning: an overview Hosts and vectors Subcloning DNA libraries Screening libraries

9、Analysis of a cloneback编辑pptPlasmid as vectorsPlasmids （质粒）（质粒）: 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 r

10、esistance to antibacterial substance.Example: ampR gene encoding the enzyme b b-lactamase which degrades penicillin antibiotics such as ampicillin; kanR for kanamycin.编辑pptbackEarlier plasmid developed编辑pptVersatile cloning plasmidPhagemid(噬菌粒）噬菌粒）编辑ppt编辑ppt编辑pptHosts and vectorsHost organism/cell:

11、where the plasmids get multiplied and propagated faithfully, 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)编辑pptGeneral features of a Vector autonomously replicati

12、ng DNA independent of hosts genome. Easily to be isolated from the host cell Most are circular, some are linear Contains at least one selective marker, which allows host cells containing the vector to be selected amongst those which do not. 1.Contains a multiple cloning site (MCS)编辑pptTypes of vecto

13、rsCloning vectorsExpression vectorsIntegration vectorsViral vectors编辑pptCloning 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: yeas

14、t artificial chromosomes (YACs)编辑pptMCSExpression 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编辑ppt编辑pptIntegration vectors: allowing th

15、e exogenous DNA to be inserted 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

16、(Agrobacterium tumefaciens Ti plasmid is used to integrate DNA into plant genome) yeast integration vectors Mammalian integration vector: gene targeting back编辑pptViral vectors:. Bacterial phage: Lambda, M13 Insect: baculoviruses Mammalian viruses: SV40, pox virus, adenovirus, retroviruses Plant viru

17、ses: TMV, PVXback编辑pptAdenoviral vector system编辑pptPlant virus vector: PVX (potato virus X)编辑ppt 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 ve

18、ctor designed to express it in a particular species. 编辑pptPreparation 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 t

19、ransformants(introduce the plasmids into host cells)Analysis of the recombinants DNA Subcloning: a flow chart Restriction endonuclease编辑pptbackAgrose Gel Electrophoresis: check your DNA at each stepSeparation and Purification of DNA fragments of interests1. Analysis of recombinant plasmidsladderRest

20、riction analysis of a plasmid编辑pptDNA 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 librariescDNA libra

21、ries编辑pptGenomic 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 cDNA library. M

22、uch more efficient in identifying a gene, but do not contain DNA coding for functional RNA or noncoding sequence.编辑pptScreening libraries Colony or plaque hybridization:Radiolabeled probes complementary to a region of the interested geneProbes: An oligonucleotide derived from the sequence of a prote

23、in product of the geneA DNA fragment/oligo from a related gene of another speciesPCR productPlating the cells carrying the library 1. Colony or plaque lift on membrane and then hybridize with the labeled probeSearching the genes of interest in a DNA library 编辑pptScreening libraries Expression screen

24、ing: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 编辑pptIdentify the protein product of an interested gen

25、e Protein activity Western blotting using a specific antibody1.In vivo expression and functional assayback编辑pptAnalysis of a clone Restriction mapping: digestion of the with restriction enzymes. Sequencing the cloned DNAbackYou may have to fully understand the function and application of all the enz

26、ymes listed in Table 1 if you want to manipulate genes编辑pptEnzymes commonly used in DNA cloning Alkaline phosphotase Reverse transcriptase, DNA ligase (T4) DNA pol I (Klenow fragment), T4, Taq Exunuclease III Mung bean nuclease and S1 nuclease Polynucleotide kinase Restriction enzymes: e.g. EcoRI, H

27、indIII RNase A, RNase H T7, T3and SP6 RNA polymerases Terminal transferase编辑pptbackG2. Preparation of plasmid DNA 1.Plasmid as vectors Plasmid minipreparation Alkaline lysis Phenol extraction Ethanol precipitation2.Cesium chloride gradient purification编辑pptPlasmid as vectorsPlasmids: small, extrachr

28、omosomal 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 enzyme b b

29、-lactamse which degrades penicillin antibiotics such as ampicillin.编辑pptPlasmid 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 denaturation, thus can be isolated

30、from the chromosomal DNA easily such as by alkaline lysis.Minipreparation (miniprep)Isolation of plasmid DNA from a few mililiters (ml) of bacterial culture. 编辑pptMiniprepsGrowth of the cells containing plasmidsCollect the cells by centrifugationAlkaline lysis resuspension alkaline lysis neutralizat

31、ionPhenol 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) 编辑pptAlkaline lysis Resuspend the

32、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 (supercoiled) and precipitation of denature

33、d proteins and chromosomal DNA.Centrifugation plasmid in supernatant (lysate)编辑pptGrow the cellHarvest the cell by centrifugationAlkaline lysis of the cellResuspend the cell pelletneutralizationPhenol extractionEthanol precipitationCsCl gradient purification编辑pptPurification of plasmid DNA by Cesium

34、 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 to DNA will unwind the DNA and reduce the DNA den

35、sitySupercoilded 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编辑pptAgarose gel electrophoresissupercoilednicked编辑pptIsolation of fragments and Agarose gel electrophore

36、sisinsertRestriction digestionAgarose gel electrophoresis3. Gel excision and purificationLigation with vector4. Transformationback编辑pptG4. Ligation, transformation and analysis of recombinantsAlkaline phosphataseDNA ligation & recombinant DNA moleculesTransformation & selection Transformatio

37、n efficiency4. Screening transformants5. Growth and storage of transformantsGel analysisFragment orientation编辑pptAlkaline phosphataseSingle restriction enzyme directed cloning Removes the phosphate groups from the 5-ends of the vector DNA linearized by a single restriction enzyme to prevent the self

38、-ligation of the vector DNA upon the followed ligation编辑pptDNA ligation Covalently join the DNA molecules with the base-pairing cohesive ends, or blunt ends, if the 5-ends have phosphate groups. 编辑ppt X if the vector is phosphorylatedRecombinant DNA molecules编辑pptThe use of alkaline phosphatase to p

39、revent religation of vector moleculesG -OHCTTAA -OH编辑pptTransformation and selectionCompetent cells (感受态细胞感受态细胞): 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）（转化）: a process of uptake of exogenous DNA by competent cells. Heat-shock （热休克）（热休克）: After the DNA is uptaken, the cells shall be put at 42C