分子生物学教学课件：2-Section G Gene manipulation-1

1、DNA 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.) DNA clonin

2、g (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, and norma

3、lly 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.Basic procedureof DNA cloningVectorGenomic fragment (restriction, PCR), cDNA (insert)Plasmid preparation

4、 (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 Isolation and manipulation of fragments of an orga

5、nisms 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 engineeringApplications of DNA cloning1.

6、Sequencing, hence to derive protein sequence; genomics2. Isolation and analysis of gene promoter etc3. Investigation of protein/enzyme/RNA function in various forms.4. Identification mutations, genetic diseases5. Biotechnology: proteins of pharmaceutical importance6. Transgenic plants and animals7.

7、Gene therapyDNA 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 technical tools of mol

8、ecular biology G1 DNA cloning: an overview (basic concepts)G2 Preparation of plasmid DNAG3Ligation, transformation and analysis of recombinants Section G Gene manipulationSection G Gene manipulationG1 DNA cloning: an overview1. Hosts and vectors2. Subcloning3. DNA libraries4. Screening libraries5. A

9、nalysis of a clonebackPlasmid 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 resista

10、nce to antibacterial substance.Example: ampR gene encoding the enzyme b b-lactamase which degrades penicillin antibiotics such as ampicillin; kanR for kanamycin.backEarlier plasmid developedVersatile cloning plasmidPhagemid(噬菌粒）噬菌粒）Hosts and vectorsHost organism/cell: where the plasmids get multipli

11、ed 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)General features of a Vector1.autonomously replicating DNA independent of hosts genome.

12、2.Easily to be isolated from the host cell3.Most are circular, some are linear4.Contains at least one selective marker, which allows host cells containing the vector to be selected amongst those which do not. 5.Contains a multiple cloning site (MCS)Types of vectorsCloning vectorsExpression vectorsIn

13、tegration vectorsViral vectorsCloning 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)MCSExpressi

14、on 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 vectorsIntegration vectors: allowing the exogenous DNA to be inserted and integrated into a chro

15、mosomal 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 plasmid is used to integrat

16、e DNA into plant genome) yeast integration vectors Mammalian integration vector: gene targeting backViral vectors:. Bacterial phage: Lambda, M13 Insect: baculoviruses Mammalian viruses: SV40, pox virus, adenovirus, retroviruses Plant viruses: TMV, PVXbackAdenoviral vector systemPlant virus vector: P

17、VX (potato virus X) Subcloning Transfer of a fragment of cloned DNA from one vector to another.1. Enables us to investigate a short region of a large cloned fragment in more detail.2. To transfer a gene from one plasmid to a vector designed to express it in a particular species. Preparation of plasm

18、ids 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(introduce the plasmids into host cells)Analysis of the recombinan

19、ts DNA Subcloning: a flow chart Restriction endonucleasebackAgrose Gel Electrophoresis: 1. check your DNA at each step2. Separation and Purification of DNA fragments of interests3. Analysis of recombinant plasmidsladderRestriction analysis of a plasmidDNA libraries are sets of DNA clones, each of wh

20、ich 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 librariesGenomic librariesprepared form random fragments of genomic DNA, which may be i

21、nefficient 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. Much more efficient in identifying a gene, but do not contain DNA coding for functional

22、RNA or noncoding sequence.Screening libraries Colony or plaque hybridization:1. Radiolabeled probes complementary to a region of the interested geneProbes: An oligonucleotide derived from the sequence of a protein product of the geneA DNA fragment/oligo from a related gene of another speciesPCR prod

23、uct2. Plating the cells carrying the library 3. Colony or plaque lift on membrane and then hybridize with the labeled probeSearching the genes of interest in a DNA library Screening libraries Expression screening:Specific antibody to gene product Screening library by PCR.:Specially prepared libraryF

24、unctional screening.:Complementation to a lethal phenotype, possible for other kinds of positive screeningSearching the genes of interest in a DNA library Identify the protein product of an interested gene1.Protein activity2.Western blotting using a specific antibody3.In vivo expression and function

25、al assaybackAnalysis of a clone 1.Restriction mapping: digestion of the with restriction enzymes.2.Sequencing the cloned DNAbackYou may have to fully understand the function and application of all the enzymes listed in Table 1 if you want to manipulate genesEnzymes commonly used in DNA cloning 1.Alk

26、aline phosphotase2.Reverse transcriptase, DNA ligase (T4)3.DNA pol I (Klenow fragment), T4, Taq4.Exunuclease III 5.Mung bean nuclease and S1 nuclease6.Polynucleotide kinase7.Restriction enzymes: e.g. EcoRI, HindIII8.RNase A, RNase H9.T7, T3and SP6 RNA polymerases10. Terminal transferasebackG2. Prepa

27、ration of plasmid DNA 1.Plasmid as vectors2.Plasmid minipreparation3.Alkaline lysis4.Phenol extraction5.Ethanol precipitation6.Cesium chloride gradient purificationPlasmid as vectorsPlasmids: small, extrachromosomal circular molecules, from 2 to 200 kb in size, which exist in multiple copies within

28、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-lactamse which degrades penicillin antibiotics such as ampicillin.Plasmid minipreparation fr

29、om 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 from the chromosomal DNA easily such as by alkaline lysis.Minipreparation (miniprep)Isolation of p

30、lasmid DNA from a few mililiters (ml) of bacterial culture. Minipreps1. Growth of the cells containing plasmids2. Collect the cells by centrifugation3. Alkaline lysis resuspension alkaline lysis neutralization4. Phenol extraction to get rid of the protein contaminants5. Ethanol precipitation to conc

31、entrate the nucleic acids remained (0.3M NaAc, 2-3 vol ethanol). 6. Resuspend in suitable buffer: TE10/1, pH 8.0 (Please note that RNase A is very bad for the lab working with RNA) Alkaline lysis Resuspend the cells in a buffer solutionLysozyme to digest the cell wall (optional)Cell lysis in lysis b

32、uffer 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 denatured proteins and chromosomal DNA.Centrifugation plasmid in supernatant (lysate)Grow the cell

33、Harvest the cell by centrifugationAlkaline lysis of the cellResuspend the cell pelletneutralizationPhenol extractionEthanol precipitationCsCl gradient purificationPurification of plasmid DNA by Cesium chloride gradient centrifugation1. CsCl gradient purification is the last step of large scale plasm

34、id DNA purification2. Laborious3. Best for the production of very pure supercoiled plasmid DNA4. The presence of ethidium bromide (EB) is important. Binding of EB 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 densi

35、tySupercoiled DNA may be purified from protein, RNA chromosomal DNA and nicked plasmid DNA in one step!backAgarose gel electrophoresissupercoilednickedIsolation of fragments and Agarose gel electrophoresisinsert1. Restriction digestion2. Agarose gel electrophoresis3. Gel excision and purification4.

36、Ligation with vector5. TransformationbackG4. Ligation, transformation and analysis of recombinants1. Alkaline phosphatase2. DNA ligation & recombinant DNA molecules3. Transformation & selection Transformation efficiency4. Screening transformants5. Growth and storage of transformants6. Gel an

37、alysisFragment orientationAlkaline 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-ligation of the vector DNA upon the followed ligationDNA ligation Covalently join the

38、DNA molecules with the base-pairing cohesive ends, or blunt ends, if the 5-ends have phosphate groups. X if the vector is phosphorylatedRecombinant DNA moleculesThe use of alkaline phosphatase to prevent religation of vector molecules G -OHCTTAA -OHTransformation and selectionCompetent cells (感受态细胞感

39、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 a