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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.),DNA cloning(Gene manipulation)To place a rela
2、tively 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 species altogether.Propagatio
3、n 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 cloning,Vector,Genomic fragment(restriction,PCR),cDNA(insert),Plasmid preparation(vector),Restriction digestion(trimming the DNA en
4、ds),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 organisms genome,Molecular analysis of proteins or other
5、interested gene products,Impossible by direct purification,DNA cloning,Impossible by direct isolation,Crucial!,Make all possible!,Gene manipulation,molecular cloning,genetic engineering,Applications of DNA cloning,Sequencing,hence to derive protein sequence;genomicsIsolation and analysis of gene pro
6、moter etcInvestigation of protein/enzyme/RNA function in various forms.Identification mutations,genetic diseasesBiotechnology:proteins of pharmaceutical importanceTransgenic plants and animalsGene therapy,DNA Cloning,Techniques&Applications,Section G:Gene manipulation(DNA cloning&subcloning,&basic t
7、echniques)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,G1 DNA cloning:an overview(basic concepts)G2 Preparation of plasmid DNAG3Ligation,transformation and analysis of recombinants,S
8、ection G Gene manipulation,G1 DNA cloning:an overview,Hosts and vectorsSubcloningDNA librariesScreening librariesAnalysis of a clone,back,Plasmid as vectors,Plasmids（质粒）:small,extrachromosomal circular molecules,from 2 to 200 kb in size,which exist in multiple copies within the host cells.contain an
9、 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-lactamase which degrades penicillin antibiotics such as ampicillin;kanR for kanamycin.,back,Earlier plasmid developed,Vers
10、atile cloning plasmid,Phagemid(噬菌粒）,Hosts and vectors,Host organism/cell: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
11、 genome),General features of a Vectorautonomously replicating DNA independent of hosts genome.Easily to be isolated 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.Contai
12、ns a multiple cloning site(MCS),Types of vectors,Cloning vectorsExpression vectorsIntegration vectorsViral vectors,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(co
13、smids).Yeast cloning vector:yeast artificial chromosomes(YACs),Expression 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,Integration vectors:
14、allowing the 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
15、 vectors(Agrobacterium tumefaciens Ti plasmid is used to integrate DNA into plant genome)yeast integration vectors Mammalian integration vector:gene targeting,back,Viral vectors:.Bacterial phage:Lambda,M13 Insect:baculoviruses Mammalian viruses:SV40,pox virus,adenovirus,retroviruses Plant viruses:TM
16、V,PVX,back,Adenoviral vector system,Plant virus vector:PVX(potato virus X),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 to express
17、 it in a particular species.,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(introduce the plasmids int
18、o host cells),Analysis of the recombinants,DNA Subcloning:a flow chart,Restriction endonuclease,back,Agrose Gel Electrophoresis:check your DNA at each stepSeparation and Purification of DNA fragments of interestsAnalysis of recombinant plasmids,ladder,Restriction analysis of a plasmid,DNA libraries,
19、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 librariescDNA libraries,Genomic librariesprepared form
20、random fragments of genomic DNA,which may be inefficient to find a gene because of the huge abundance of the non-coding DNA,cDNA 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,bu
21、t do not contain DNA coding for functional RNA or noncoding sequence.,Screening libraries,Colony or plaque hybridization: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
22、 related gene of another speciesPCR productPlating the cells carrying the library Colony or plaque lift on membrane and then hybridize with the labeled probe,Searching the genes of interest in a DNA library,Screening libraries,Expression screening:Specific antibody to gene product Screening library
23、by PCR.:Specially prepared libraryFunctional screening.:Complementation to a lethal phenotype,possible for other kinds of positive screening,Searching the genes of interest in a DNA library,Identify the protein product of an interested geneProtein activityWestern blotting using a specific antibodyIn
24、 vivo expression and functional assay,back,Analysis of a clone,Restriction mapping:digestion of the with restriction enzymes.Sequencing the cloned DNA,back,You may have to fully understand the function and application of all the enzymes listed in Table 1 if you want to manipulate genes,Enzymes commo
25、nly used in DNA cloning,Alkaline phosphotaseReverse transcriptase,DNA ligase(T4)DNA pol I(Klenow fragment),T4,TaqExunuclease III Mung bean nuclease and S1 nucleasePolynucleotide kinaseRestriction enzymes:e.g.EcoRI,HindIIIRNase A,RNase HT7,T3and SP6 RNA polymerases Terminal transferase,back,G2.Prepar
26、ation of plasmid DNA,1.Plasmid as vectorsPlasmid minipreparationAlkaline lysisPhenol extractionEthanol precipitationCesium chloride gradient purification,Plasmid as vectors,Plasmids:small,extrachromosomal circular molecules,from 2 to 200 kb in size,which exist in multiple copies within the host cell
27、s.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-lactamse which degrades penicillin antibiotics such as ampicillin.,Plasmid minipreparation from E.coli,Plasmid
28、s2-20 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 plasmid DNA from a few
29、mililiters(ml)of bacterial culture.,Minipreps,Growth of the cells containing plasmidsCollect the cells by centrifugationAlkaline lysis resuspension alkaline lysis neutralizationPhenol extraction to get rid of the protein contaminantsEthanol precipitation to concentrate the nucleic acids remained(0.3
30、M 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),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
31、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,Harvest the cell by centrifugation,Alkaline lysis of t
32、he cell,Resuspend the cell pellet,neutralization,Phenol extraction,Ethanol precipitation,CsCl gradient purification,Purification of plasmid DNA by Cesium chloride gradient centrifugation,CsCl gradient purification is the last step of large scale plasmid DNA purificationLaboriousBest for the producti
33、on 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 densitySupercoilded DNA bind less EB than linear DNA or nicked DNA,thus has a higher density,Supercoiled DNA may be purified from protein,RNA chromosoma
34、l DNA and nicked plasmid DNA in one step!,back,Agarose gel electrophoresis,supercoiled,nicked,Isolation of fragments and Agarose gel electrophoresis,insert,Restriction digestionAgarose gel electrophoresis,3.Gel excision and purificationLigation with vectorTransformation,back,G4.Ligation,transformati
35、on and analysis of recombinants,Alkaline phosphataseDNA ligation&recombinant DNA moleculesTransformation&selection Transformation efficiency4.Screening transformants5.Growth and storage of transformantsGel analysisFragment orientation,Alkaline phosphataseSingle restriction enzyme directed cloning Re
36、moves 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 ligation,DNA ligation Covalently join the DNA molecules with the base-pairing cohesive ends,or blunt ends,if the 5-ends have phospha
37、te groups.,X if the vector is phosphorylated,Recombinant DNA molecules,The use of alkaline phosphatase to prevent religation of vector molecules,G-OHCTTAA-OH,Transformation and selection,Competent cells(感受态细胞):E.coli cells treated with Ca2+solution are susceptible to take up exogenous DNA.Enzymes in
38、volved 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 in order to increase the transformation efficiency,Select
39、ion with antibiotic resistance(ampr),Transformation efficiency:number of colonies formed per microgram(mg)of input DNA.Ranges from 103 to more than 108.105 is adequate for a simple cloning.Electroporation（电击穿）is more efficient,up to 109.,Growth and storage of transformants（转化子）,Can be grow in liquid
40、 broth or solid platesMaintain the selection pressure by the presence of the corresponding antibiotics.Store the transformant bacteria by freezing a portion in the presence of 15%glycerol to protect from ice crystal formation,Gel analysis and fragment orientation,Distinguish the recombinant plasmids
41、 from the original vectors bySize of the plasmids(not work well if the plasmids are prepared with alkaline lysis)Restriction digestion2.Determine the orientation of an inserted DNA fragment cloned by a single enzyme by restriction digestion that cuts asymmetrically within the insert sequence,and once at a specific site of the vector,3.Analysis of a clone by restriction mapping,back,Summary:Hosts and vectors,basic cloning,library and screening,analysis of a clone.Preparation of plasmidsRestriction enzymes and agarose gel electrophoresis.Ligation,transformants and clone analysis,
