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Section G Gene manipulation Molecular Biology Content G1 DNA CLONING: AN OVERVIEW G2 PREPARATION OF PLASMID DNA G3 RESTRICTION ENZYMES AND ELECTROPHORESIS.

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Presentation on theme: "Section G Gene manipulation Molecular Biology Content G1 DNA CLONING: AN OVERVIEW G2 PREPARATION OF PLASMID DNA G3 RESTRICTION ENZYMES AND ELECTROPHORESIS."— Presentation transcript:

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2 Section G Gene manipulation Molecular Biology

3 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

4 G1-1 DNA cloning G1-2 Hosts and vectors G1-3 Subcloning G1-4 DNA libraries G1-5 Screening libraries G1-6 Analysis of a clone G1 DNA cloning: an overview Molecular Biology

5 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

6 G1-1 DNA cloning Molecular Biology G1 DNA cloning: An Overview

7 G1-2 Hosts and vectors Most 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. Molecular Biology G1 DNA cloning: An Overview

8 General features of a Vector 1.autonomously replicating DNA 2.selective marker 3.multiple cloning site (MCS) G1 DNA cloning: An Overview Molecular Biology

9 Types of vectors A:Cloning vectors B:Expression vectors C:Integration vectors G1 DNA cloning: An Overview Molecular Biology

10 Cloning vectors E. coli cloning vector Yeast cloning vector(YACS) Molecular Biology

11 MCS Expression 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

12 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

13 G1-3 Subcloning G1 DNA cloning: An Overview Molecular Biology

14 Agrose Gel Electrophoresis: 1.check your DNA at each step 2.Separation and Purification of DNA fragments of interests 3.Analysis of recombinant plasmids Marker Restriction analysis of a plasmid G1 DNA cloning: An Overview Molecular Biology

15 G1-4 DNA libraries G1 DNA cloning: An Overview Genomic libraries prepared 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

16 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

17 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

18 G1 DNA cloning: An Overview Molecular Biology

19 G2 Preparation of plasmid DNA. Plasmid as vector. Plasmid minipreparation. Alkaline lysis. Phenol extraction. Ethanol precipitation. Cesium chloride gradient (purification) Gene manipulation Molecular Biology

20 G2-2 Plasmid minipreparation from E. coli Plasmids ~2-20 kb in length that much smaller than E. coli chromosomal 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 bacterial culture. G2 Preparation of plasmid DNA Molecular Biology

21 Miniprep 1.Growth of the cells containing plasmids 2.Collect the cells by centrifugation 3.Alkaline lysis 4.Phenol extraction to get rid of the protein contaminants 5.Ethanol precipitation to concentrate the nucleic acids remained. G2 Preparation of plasmid DNA Molecular Biology

22 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): 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. G2 Preparation of plasmid DNA Molecular Biology

23 Fig. 1. Plasmid preparation Molecular Biology

24 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

25 G3 Restriction Enzymes and electrophoresis.Restriction endonuclease. Recognition sequences. Cohesive ends. Restriction digests. Agarose gel electrophoresis. Isolation of fragments Gene manipulation Molecular Biology

26 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

27 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

28 Recognition sequences 1.Recognize 4-8 bp. Most recognition sequences are 6 bp which occurs at a rate of 4 6 =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

29 G3-4 Restriction digestion G3 Restriction enzymes and electrophoresis Fig. 2. The digestion of a plasmid with two different restriction enzymes. Molecular Biology

30 G3-5 Agrose gel electrophoresis Agrose: 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

31 Agrose gel electrophoresis G3 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

32 G4 Ligation, transformation and analysis of recombinants G4.1 Alkaline phophatse G4.2-3 DNA ligation & recombinant DNA molecules G4.4-5 Transformation & selection G4.6 Transformation efficiency G4.7Screening transformants G4.8 Growth and storage of transformants G4.9 Gel analysis G4.10 Fragment orientation Gene manipulation Molecular Biology

33 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

34 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

35 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 42 o C for 1 min in order to induce the suppressed enzymes for cell defending G4 Ligation, transformation and analysis of recombinants Molecular Biology

36 Selection with antibiotic resistance (amp r ) Transformantion efficiency: number of colonies formed per microgram (mg) of input DNA. Ranges from 10 3 to more than is adequate for a simple cloning. G4 Ligation, transformation and analysis of recombinants Molecular Biology

37 Transformantion efficiency: number of colonies formed per microgram (mg) of input DNA. Ranges from 10 3 to more than is adequate for a simple cloning. G4-6 Transformation efficiency G4 Ligation, transformation and analysis of recombinants Molecular Biology

38 G4 Ligation, transformation and analysis of recombinants Fig. 4. The analysis of recombinant plasmids by agarose gel electrophoresis Molecular Biology


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