Presentation on theme: "Section G Gene manipulation"— Presentation transcript:
1 Section G Gene manipulation Molecular Biology CourseSection G Gene manipulation
2 G1-5 Screening libraries G1-6 Analysis of a clone Gene manipulationG1-1 DNA cloningG1-2 Hosts and vectorsG1-3 SubcloningG1-4 DNA librariesG1-5 Screening librariesG1-6 Analysis of a clone
3 G1 DNA cloning: an overview Gene manipulationG1 DNA cloning: an overview
4 G1-1 DNA cloning (definition) G1 DNA cloning: An OverviewG1-1 DNA cloning (definition)DNA cloning is 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, forming recombinant DNA, which can be replicates independently of the original genome, 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.
5 G1 DNA cloning: An Overview G1-2 Hosts and vectorsHost 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 genome)
6 General features of a Vector G1 DNA cloning: An OverviewGeneral features of a Vectorautonomously replicating DNA independent of host’s 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.Contains a multiple cloning site (MCS)
7 Types of vectors Cloning vectors Expression vectors G1 DNA cloning: An OverviewTypes of vectorsCloning vectorsExpression vectorsIntegration vectors
8 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)
9 G1 DNA cloning: An Overview Expression vectors: allowing the exogenous DNA to be inserted and expressed. Promoter and terminator for RNA transcription are required.bacterial expression vectorsyeast expression vectorsmammalian expression vectorsMCS
10 G1 DNA cloning: An Overview Integration vectors: allowing the exogenous DNA to be inserted and integrated into a chromosomal DNA after a transformation. The integration is 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 integrate DNA into plant genome)yeast integration vectorsMammalian integration vector: virus based
11 G1 DNA cloning: An Overview G1-3 SubcloningTransfer 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 it in a particular species.
12 DNA Subcloning: a flow chart Preparation of plasmids containing a cloned DNA fragment (insert)Plasmid preparation(vector)Restriction digestion(trimming the DNA ends)RestrictionendonucleaseLigation(join the insert and the vector)Transformation & selection of transformants(introduce the plasmids into host cells)Assay of the recombinants
13 G1 DNA cloning: An Overview Agrose Gel Electrophoresis: check your DNA at each stepSeparation and Purification of DNA fragments of interestsAnalysis of recombinant plasmidsladderRestriction analysis of a plasmid
14 G1 DNA cloning: An Overview G1-4 DNA librariesDNA 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 libraries cDNA libraries
15 Genomic libraries cDNA libraries G1 DNA cloning: An Overview prepared form random fragments of genomic DNA, which may be inefficient to find a gene because of the huge abundance of the non-coding DNAcDNA librariesDNA 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 functional RNA or noncoding sequence.
16 G1-5 Screening libraries G1 DNA cloning: An OverviewG1-5 Screening librariesSearching the genes of interest in a DNA libraryHybridization to identify the interested DNA or its RNA productRadiolabeled probes which is 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 speciesBlotting the DNA or RNA on a membraneHybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane
17 Identify the protein product of an interested gene G1 DNA cloning: An OverviewIdentify the protein product of an interested geneProtein activityWestern blotting using a specific antibody
18 G1 DNA cloning: An Overview G1-6 Analysis of a cloneRestriction mapping: digestion of the with restriction enzymes.Sequencing the cloned DNAYou may have to fully understand the function and application of all the enzymes listed in Table 1 before the final exam
19 G2 Preparation of plasmid DNA Gene manipulationG2 Preparation of plasmid DNAG2-1 Plasmid as vectorsG2-2 Plasmid minipreparationG2-3 Alkaline lysisG2-4 Phenol extractionG2-5 Ethanol precipitationG2-6 Cesium chloride gradient (purification)
20 G2 Preparation of plasmid DNA G2-1 Plasmid as vectorsPlasmids: 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 gene encoding the enzyme b-lactamse which degrades penicillin antibiotics such as ampicillin.
21 G2-2 Plasmid minipreparation from E. coli G2 Preparation of plasmid DNAG2-2 Plasmid minipreparation from E. coliPlasmids~2-20 kb in length that 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 alkaline lysis.Minipreparation (miniprep)Isolation of plasmid DNA from a few mililiters (ml) of bacterial culture.
22 Miniprep G2 Preparation of plasmid DNA Growth of the cells containing plasmidsCollect the cells by centrifugationAlkaline lysisresuspension alkaline lysis neutralizationPhenol extraction to get rid of the protein contaminantsEthanol precipitation to concentrate the nucleic acids remained. (Please noted that RNase A is very bad for the lab working with RNA)
23 G2-3 Alkaline lysis G2 Preparation of plasmid DNA 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 (supercoiled) and precipitation of denatured proteins and chromosomal DNA which can not be renatured because of its size and physical property of easily being sheared.
24 Grow the cellHarvest the cell by centrifugationResuspend the cell pelletAlkaline lysis of the cellneutralizationPhenol extractionEthanol precipitationCsCl gradient purification
25 G2- 6 Cesium chloride gradient centrifugation G2 Preparation of plasmid DNAG2- 6 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 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!!
26 G3 Restriction Enzymes and electrophoresis Gene manipulationG3 Restriction Enzymes and electrophoresisG3-1 Restriction endonucleaseG3-2 Recognition sequencesG3-3 Cohesive endsG3-4 Restriction digestsG3-5 Agarose gel electrophoresisG3-6 Isolation of fragments
27 G3-1 Restriction endonuclease G3 Restriction enzymes and electrophoresisG3-1 Restriction endonucleaseBacterial enzymes which cut DNA into defined and reproducible fragmentsIdentified in the 1960sKey discovery which allowed the DNA cloning to become a reality
28 G3 Restriction enzymes and electrophoresis One component of the bacterial restriction-modification system, a natural defense mechanism of bacteria to against the introduction of foreign DNA into the cellRestriction endonuclease: recognize a short, symmetrical DNA sequence, and cut DNA backbone in each strand at a specific site within that sequence (kill foreign DNA)Mythylase: methylates C or A of the cellular DNA
30 Recognition sequences G3 Restriction enzymes and electrophoresisRecognition sequencesRecognize 4-8 bp. Most recognition sequences are 6 bp which occurs at a rate of 46=4096 bp.Highly specificRestriction enzymesCommercially availableRequire Mg2+ for enzymatic activity
31 G3-4 Restriction digestion G3 Restriction enzymes and electrophoresisG3-4 Restrictiondigestion
32 G3-5 Agrose gel electrophoresis G3 Restriction enzymes and electrophoresisG3-5 Agrose gel electrophoresisAgrose: a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution (0.5%-3%)Negatively charged DNA- ve electrode+ ve electrode
33 Agrose gel electrophoresis G3 Restriction enzymes and electrophoresisAgrose gel electrophoresis
34 Isolation of fragments and Agarose gel electrophoresis G3 Restriction enzymes and electrophoresisIsolation of fragments andAgarose gel electrophoresisRestriction digestionAgarose gel electrophoresisinsert3. Gel excision and purificationLigation with vectortransformation
35 G4 Ligation, transformation and analysis of recombinants Gene manipulationG4 Ligation, transformation and analysis of recombinantsG4-1 Alkaline phophatseG4-2&3 DNA ligation & recombinant DNA moleculesG4-4&5 Transformation & selectionG4-6 Transformation efficiencyG4-7 Screening transformantsG4-8 Growth and storage of transformantsG4-9 Gel analysisG4-10 Fragment orientation
36 G4-1 Alkaline phophatse Single restriction enzyme directed cloning G4 Ligation, transformation and analysis of recombinantsG4-1 Alkaline phophatseremoves the phosphate groups from th 5’-ends of the vector DNA linearized by a single restriction enzyme to prevent the self-ligation of the vector DNA upon the followed ligationSingle restriction enzyme directed cloning
37 G4-2&3 DNA ligation & recombinant DNA molecules G4 Ligation, transformation and analysis of recombinantsG4-2&3 DNA ligation & recombinant DNA moleculesDNA ligationCovalently join the DNA molecules with the base-pairing cohesive ends, or blunt ends, if the 5’-ends have phosphate groups.
38 Recombinant DNA molecules G4 Ligation, transformation and analysis of recombinantsRecombinant DNA moleculesXif the vector is phosphoralated
39 G4 Ligation, transformation and analysis of recombinants The use of alkaline phosphate to prevent religation of vector molecules
40 G4-4&5 Transformation and selection G4 Ligation, transformation and analysis of recombinantsG4-4&5 Transformation and selectionCompetent cells: 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: a process of uptake of exogenous DNA by competent cells.Heat-shock: After the DNA is uptaken, the cells shall be put at 42oC for 1 min in order to induce the suppressed enzymes for cell defending
41 G4 Ligation, transformation and analysis of recombinants Selection with antibiotic resistance (ampr)Transformantion efficiency: number of colonies formed per microgram (mg) of input DNA. Ranges from 103 to more than is adequate for a simple cloning.
42 G4-6 Transformation efficiency G4 Ligation, transformation and analysis of recombinantsG4-6 Transformation efficiencyTransformantion efficiency: number of colonies formed per microgram (mg) of input DNA. Ranges from 103 to more than is adequate for a simple cloning.
43 G4-7 Growth and storage of transformants G4 Ligation, transformation and analysis of recombinantsG4-7 Growth and storage of transformantsCan be grow in liquid broth or solid platesMaintain the selection pressure by the presence of the corresponding antibiotics. Plasmid stability and loseStore the transformant bacteria by freezing a portion in the presence of glycerol to protect from ice crystal formation
44 Distinguish the recombinant plasmids from the recreated vectors by G4 Ligation, transformation and analysis of recombinantsG4-8&9 Gel analysis and fragment orientationDistinguish the recombinant plasmids from the recreated vectors bySize of the plasmids (not work well if the plasmids are prepared with alkaline lysis)Restriction digestion2. Determine the orientation of a 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
45 G4 Ligation, transformation and analysis of recombinants
46 G4 Ligation, transformation and analysis of recombinants 3. Analysis of a clone by restriction mapping