Presentation on theme: "Section G Gene manipulation"— Presentation transcript:
1 Section G Gene manipulation Molecular BiologySection G Gene manipulation
2 Content G1 DNA CLONING: AN OVERVIEW G2 PREPARATION OF PLASMID DNA Molecular BiologyContentG1 DNA CLONING: AN OVERVIEWG2 PREPARATION OF PLASMID DNAG3 RESTRICTION ENZYMES AND ELECTROPHORESISG4 LIGATION, TRANSFORMATION AND ANALYSIS OF RECOMBINANTS
3 G1 DNA cloning: an overview Molecular BiologyG1 DNA cloning: an overviewG1-1 DNA cloningG1-2 Hosts and vectorsG1-3 SubcloningG1-4 DNA librariesG1-5 Screening librariesG1-6 Analysis of a clone
4 Molecular BiologyG1 DNA cloning: An OverviewG1-1 DNA cloningThe transfer of a DNA fragment of interest from one organism to a self-replicating genetic element such as a bacterial plasmid. The DNA of interest can then be propagated in a foreign host cell. This technology has been around since the 1970s, and it has become a common practice in molecular biology labs today.
5 Molecular BiologyG1 DNA cloning: An OverviewG1-1 DNA cloning
6 Molecular BiologyG1 DNA cloning: An OverviewG1-2 Hosts and vectorsMost of the routine manipulations involved in gene cloning use E.coli as the host ogranism. Plasmids and bacteriophages may be used as cloning vectors in E.coli. Vectors based on plasmids, viruses and whole chromosomes have been used to carry foreign genes into other prokaryotic and eukaryotic organiams.
7 General features of a Vector Molecular BiologyG1 DNA cloning: An OverviewGeneral features of a Vectorautonomously replicating DNAselective markermultiple cloning site (MCS)
8 Types of vectors A:Cloning vectors B:Expression vectors Molecular BiologyG1 DNA cloning: An OverviewTypes of vectorsA:Cloning vectorsB:Expression vectorsC:Integration vectors
10 G1 DNA cloning: An Overview Molecular BiologyG1 DNA cloning: An OverviewMCSExpression vectors: allowing the exogenous DNA to be inserted and expressed. Promoter and terminator for RNA transcription are required.. bacterial expressionvectors. yeast expression vectors. mammalian expression vectors
11 G1 DNA cloning: An Overview Molecular BiologyG1 DNA cloning: An OverviewIntegration vectors: allowing the exogenous DNA to be inserted and integrated into a chromosomal DNA after a transformation.
12 Molecular BiologyG1 DNA cloning: An OverviewG1-3 Subcloning
13 G1 DNA cloning: An Overview Molecular BiologyG1 DNA cloning: An OverviewAgrose Gel Electrophoresis:check your DNA at each stepSeparation and Purification of DNA fragments of interestsAnalysis of recombinant plasmidsMarkerRestriction analysis of a plasmid
14 G1-4 DNA libraries G1 DNA cloning: An Overview Genomic libraries Molecular BiologyG1 DNA cloning: An OverviewG1-4 DNA librariesGenomic 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 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.
15 G1-5 Screening libraries Molecular BiologyG1 DNA cloning: An OverviewG1-5 Screening librariesSearching the genes of interest in a DNA libraryLibraries are screened for the presence of a gene sequence by hybridization with a sequence derived from its protein product or a rlated gene, or through the screening of the protein products of the cloned fragments.
16 Molecular BiologyG1 DNA cloning: An OverviewG1-6 Analysis of a cloneOnce identified, a cloned gene may be analysed by restriction mapping, and ultimatedly DNA sequencing, beforebeing used in any of the diverse applications of DNA cloning.Restriction mapping: digestion of the with restriction enzymes.Sequencing the cloned DNA
17 G1 DNA cloning: An Overview Molecular BiologyG1 DNA cloning: An Overview
18 G2 Preparation of plasmid DNA Molecular BiologyGene manipulationG2 Preparation of plasmid DNA. Plasmid as vector. Plasmid minipreparation. Alkaline lysis. Phenol extraction. Ethanol precipitation. Cesium chloride gradient (purification)
19 G2-2 Plasmid minipreparation from E. coli Molecular BiologyG2 Preparation of plasmid DNAG2-2 Plasmid minipreparationfrom 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.
20 Miniprep G2 Preparation of plasmid DNA Molecular BiologyG2 Preparation of plasmid DNAMiniprepGrowth of the cells containing plasmidsCollect the cells by centrifugationAlkaline lysisPhenol extraction to get rid of the protein contaminantsEthanol precipitation to concentrate the nucleic acids remained.
21 G2-3 Alkaline lysis G2 Preparation of plasmid DNA Molecular BiologyG2 Preparation of plasmid DNAG2-3 Alkaline lysisResuspend the cells in a buffer solutionLysozyme to digest the cell wallCell lysis in lysis buffer containing SDS and NaOHNeutralization buffer containing KOAc (pH 5): renaturation of plasmid DNA and precipitation of denatured proteins and chromosomal DNA which can not be renatured because of its size and physical property of easily being sheared.
23 G2- 6 Cesium chloride gradient centrifugation Molecular BiologyG2 Preparation of plasmid DNAG2- 6 Cesium chloride gradient centrifugationA CsCl gradient can be used as part of a large-scale plasmid preparation to purify supercoiled plasmid DNA away from protein, Rna and linear or nicked DNA.
24 G3 Restriction Enzymes and electrophoresis Molecular BiologyGene manipulationG3 Restriction Enzymes and electrophoresis.Restriction endonuclease. Recognition sequences. Cohesive ends. Restriction digests. Agarose gel electrophoresis. Isolation of fragments
25 G3-1 Restriction endonuclease Molecular BiologyG3 Restriction enzymes and electrophoresisG3-1 Restriction endonucleaseRestriction enonucleases are bacterial enzymes which cut(hydrolyze) DNA into defined and reproducible fragments. In bacteria, they form part of the restriction-modification defense mechanism against foreign DNA. They are the basic tools of gene cloning.
26 G3-2&3 Restriction sequences&Cohesive ends Molecular BiologyG3 Restriction enzymes and electrophoresisG3-2&3 Restriction sequences&Cohesive endsFig. 1. (a) The action of restriction endonucleases at their recognition sequences; (b) the annealing of cohesive ends.
27 Recognition sequences Molecular BiologyG3 Restriction enzymes and electrophoresisRecognition sequencesRecognition enzymes cleave DNA symmertrically in both strands at short Palindromic(symmetrical) recognition sequences to leave a 5’-phosphate and a 3’-OH. They leave blunt ends, or protruding 5’- or 3’- termini.Recognize 4-8 bp. Most recognition sequences are 6 bp which occurs at a rate of 46=4096 bp.Highly specific
28 G3-4 Restriction digestion Molecular BiologyG3 Restriction enzymes and electrophoresisG3-4 Restriction digestionFig. 2. The digestion of a plasmid with two different restriction enzymes.
29 G3-5 Agrose gel electrophoresis Molecular BiologyG3 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
30 Agrose gel electrophoresis Molecular BiologyG3 Restriction enzymes and electrophoresisAgrose gel electrophoresisFig. 4. (a) An agarose gel of DNA restriction fragments (see text for details); (b) a calibration curve of migration distance against fragment size.
31 G4 Ligation, transformation and analysis of recombinants Molecular BiologyGene 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
32 G4 Ligation, transformation and analysis of recombinants Molecular BiologyG4 Ligation, transformation and analysis of recombinantsFig. 1. The ligation of vector and target to yield recombinant and nonrecombinant productsXif the vector is phosphoralated
33 G4 Ligation, transformation and analysis of recombinants Molecular BiologyG4 Ligation, transformation and analysis of recombinantsThe use of alkaline phosphate to prevent religation of vector moleculesFig. 2. The use of alkaline phosphatase to prevent religation of vector molecules.
34 G4.4-5 Transformation and selection Molecular BiologyG4 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
35 G4 Ligation, transformation and analysis of recombinants Molecular BiologyG4 Ligation, transformation and analysis of recombinantsSelection 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.
36 G4-6 Transformation efficiency Molecular BiologyG4 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.
37 G4 Ligation, transformation and analysis of recombinants Molecular BiologyG4 Ligation, transformation and analysis of recombinantsFig. 4. The analysis of recombinant plasmids by agarose gel electrophoresis