section g gene manipulation molecular biology content g1 dna cloning: an overview g2 preparation of...
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Section G Gene manipulation
Molecular Biology
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Content
G1 DNA CLONING: AN OVERVIEW G2 PREPARATION OF PLASMID DNA G3 RESTRICTION ENZYMES AND
ELECTROPHORESIS G4 LIGATION, TRANSFORMATION AND
ANALYSIS OF RECOMBINANTS
Molecular Biology
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G1-1 DNA cloningG1-2 Hosts and vectorsG1-3 SubcloningG1-4 DNA librariesG1-5 Screening librariesG1-6 Analysis of a clone
G1 DNA cloning: an overview
Molecular Biology
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G1-1 DNA cloning
The 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.
Molecular Biology
G1 DNA cloning: An Overview
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G1-1 DNA cloning
Molecular Biology
G1 DNA cloning: An Overview
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G1-2 Hosts and vectors• Most of the routine manipulations involved i
n 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.
Molecular Biology
G1 DNA cloning: An Overview
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General features of a Vector
1.autonomously replicating DNA
2.selective marker3.multiple cloning site (MCS)
G1 DNA cloning: An Overview
Molecular Biology
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Types of vectors
A:Cloning vectors
B:Expression vectors
C:Integration vectors
G1 DNA cloning: An Overview
Molecular Biology
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Cloning vectors E. coli cloning vector Yeast cloning vector(YACS)
Molecular Biology
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MCSExpression 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
G1 DNA cloning: An Overview
Molecular Biology
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Integration vectors: allowing the exogenous DNA to be inserted and integrated into a chromosomal DNA after a transformation.
G1 DNA cloning: An Overview
Molecular Biology
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G1-3 Subcloning
G1 DNA cloning: An Overview
Molecular Biology
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Agrose Gel Electrophoresis: 1. check your DNA at each step2. Separation and Purification of DNA fragments of
interests3. Analysis of recombinant plasmids M
arke
r
Restriction analysis of a plasmid
G1 DNA cloning: An Overview
Molecular Biology
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G1-4 DNA libraries G1 DNA cloning: An Overview
Genomic librariesprepared form 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, but do not contain DNA coding functional RNA or noncoding sequence.
Molecular Biology
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G1-5 Screening libraries
Libraries 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.
Searching the genes of interest in a DNA library
G1 DNA cloning: An Overview
Molecular Biology
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G1-6 Analysis of a clone
1.Restriction mapping: digestion of the with restriction enzymes.
2.Sequencing the cloned DNA
G1 DNA cloning: An Overview
Once 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.
Molecular Biology
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G1 DNA cloning: An Overview
Molecular Biology
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G2 Preparation of plasmid DNA
. Plasmid as vector
. Plasmid minipreparation
. Alkaline lysis
. Phenol extraction
. Ethanol precipitation
. Cesium chloride gradient (purification)
Gene manipulationMolecular Biology
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G2-2 Plasmid minipreparation from E. coli
Plasmids~2-20 kb in length that much smaller than E. coli chrom
osomal DNA (4600 kb), and independently supercoiled
• Resistant 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 ba
cterial culture.
G2 Preparation of plasmid DNA
Molecular Biology
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Miniprep
1. Growth of the cells containing plasmids2. Collect the cells by centrifugation3. Alkaline lysis 4. Phenol extraction to get rid of the protein co
ntaminants5. Ethanol precipitation to concentrate the nucl
eic acids remained.
G2 Preparation of plasmid DNA
Molecular Biology
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G2-3 Alkaline lysis • Resuspend the cells in a buffer solution• Lysozyme to digest the cell wall • Cell lysis in lysis buffer containing SDS and NaOH • Neutralization buffer containing KOAc (pH 5): ren
aturation 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.
G2 Preparation of plasmid DNA
Molecular Biology
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Fig. 1. Plasmid preparation
Molecular Biology
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G2- 6 Cesium chloride gradient centrifugation
A 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.
G2 Preparation of plasmid DNA
Molecular Biology
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G3 Restriction Enzymes and electrophoresis
.Restriction endonuclease
. Recognition sequences
. Cohesive ends
. Restriction digests
. Agarose gel electrophoresis
. Isolation of fragments
Gene manipulationMolecular Biology
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G3-1 Restriction endonuclease
Restriction 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.
G3 Restriction enzymes and electrophoresis
Molecular Biology
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G3-2&3 Restriction sequences&Cohesive ends
G3 Restriction enzymes and electrophoresis
Fig. 1. (a) The action of restriction endonucleases at their recognition sequences; (b) the annealing of cohesive ends.
Molecular Biology
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Recognition sequences
1. Recognize 4-8 bp. Most recognition sequences are 6 bp which occurs at a rate of 46=4096 bp.
2. Highly specific
G3 Restriction enzymes and electrophoresis
Recognition 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.
Molecular Biology
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G3-4 Restriction digestionG3 Restriction enzymes and electrophoresis
Fig. 2. The digestion of a plasmid with two different restriction enzymes.
Molecular Biology
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G3-5 Agrose gel electrophoresisAgrose: a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution (0.5%-3%)
- ve electrode + ve electrode
Negatively charged DNA
G3 Restriction enzymes and electrophoresis
Molecular Biology
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Agrose gel electrophoresisG3 Restriction enzymes and electrophoresis
Fig. 4. (a) An agarose gel of DNA restriction fragments (see text for details); (b) a calibration curve of migration distance against fragment size.
Molecular Biology
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G4 Ligation, transformation and analysis of recombinants
G4.1 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
Gene manipulationMolecular Biology
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G4 Ligation, transformation and analysis of recombinants
X if the vector is phosphoralated
Fig. 1. The ligation of vector and target to yield recombinant and nonrecombinant products
Molecular Biology
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The use of alkaline phosphate to prevent religation of vector molecules
G4 Ligation, transformation and analysis of recombinants
Fig. 2. The use of alkaline phosphatase to prevent religation of vector molecules.
Molecular Biology
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G4.4-5 Transformation and selection
Competent 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
G4 Ligation, transformation and analysis of recombinants
Molecular Biology
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Selection with antibiotic resistance (ampr)
Transformantion 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.
G4 Ligation, transformation and analysis of recombinants
Molecular Biology
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Transformantion 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.
G4-6 Transformation efficiency
G4 Ligation, transformation and analysis of recombinants
Molecular Biology
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G4 Ligation, transformation and analysis of recombinants
Fig. 4. The analysis of recombinant plasmids by agarose gel electrophoresis
Molecular Biology