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Section G Gene manipulation Molecular Biology Course.

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Presentation on theme: "Section G Gene manipulation Molecular Biology Course."— Presentation transcript:

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2 Section G Gene manipulation Molecular Biology Course

3 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 Gene manipulation

4 G1 DNA cloning: an overview Gene manipulation

5 G1-1 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. G1 DNA cloning: An Overview

6 G1-2 Hosts and vectors Host organism/cell: where the plasmids get multiplied and propagated faithfully, which is crucial for DNA cloning. Hosts for DNA cloning vector Prokaryotic host : E. coli ( most cases) Eukaryotic host : Yeast Saccharomyces cerevisiae (large fragments of human genome) G1 DNA cloning: An Overview

7 General features of a Vector 1.autonomously replicating DNA independent of host’s genome. 2.Easily to be isolated from the host cell 3.Most are circular, some are linear 4.Contains at least one selective marker, which allows host cells containing the vector to be selected amongst those which do not. 5.Contains a multiple cloning site (MCS) G1 DNA cloning: An Overview

8 Types of vectors Cloning vectors Expression vectors Integration vectors G1 DNA cloning: An Overview

9 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)

10 MCS 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 G1 DNA cloning: An Overview

11 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 vectors Mammalian integration vector: virus based G1 DNA cloning: An Overview

12 G1-3 Subcloning Transfer of a fragment of cloned DNA from one vector to another. 1.Enables us to investigate a short region of a large cloned fragment in more detail. 2.To transfer a gene from one plasmid to a vector designed to express it in a particular species. G1 DNA cloning: An Overview

13 Preparation of plasmids containing a cloned DNA fragment (insert) Plasmid preparation (vector) Restriction digestion (trimming the DNA ends) Ligation (join the insert and the vector) Transformation & selection of transformants (introduce the plasmids into host cells) Assay of the recombinants DNA Subcloning: a flow chart Restriction endonuclease

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 ladder Restriction analysis of a plasmid G1 DNA cloning: An Overview

15 G1-4 DNA libraries 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 individuals Genomic librariescDNA libraries G1 DNA cloning: An Overview

16 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. G1 DNA cloning: An Overview

17 G1-5 Screening libraries Hybridization to identify the interested DNA or its RNA product 1.Radiolabeled probes which is complementary to a region of the interested gene Probes: An oligonucleotide derived from the sequence of a protein product of the gene A DNA fragment/oligo from a related gene of another species 2.Blotting the DNA or RNA on a membrane 3.Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane Searching the genes of interest in a DNA library G1 DNA cloning: An Overview

18 Identify the protein product of an interested gene 1.Protein activity 2.Western blotting using a specific antibody G1 DNA cloning: An Overview

19 G1-6 Analysis of a clone 1.Restriction mapping: digestion of the with restriction enzymes. 2.Sequencing the cloned DNA You may have to fully understand the function and application of all the enzymes listed in Table 1 before the final exam G1 DNA cloning: An Overview

20 G2 Preparation of plasmid DNA G2-1 Plasmid as vectors G2-2 Plasmid minipreparation G2-3 Alkaline lysis G2-4 Phenol extraction G2-5 Ethanol precipitation G2-6 Cesium chloride gradient (purification) Gene manipulation

21 G2-1 Plasmid as vectors Plasmids: 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 independently Usually carry a few genes, one of which may confer resistance to antibacterial substance. Example: amp r gene encoding the enzyme b- lactamse which degrades penicillin antibiotics such as ampicillin. G2 Preparation of plasmid DNA

22 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

23 Miniprep 1.Growth of the cells containing plasmids 2.Collect the cells by centrifugation 3.Alkaline lysis resuspension  alkaline lysis  neutralization 4.Phenol extraction to get rid of the protein contaminants 5.Ethanol precipitation to concentrate the nucleic acids remained. (Please noted that RNase A is very bad for the lab working with RNA) G2 Preparation of plasmid DNA

24 G2-3 Alkaline lysis Resuspend the cells in a buffer solution Lysozyme 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. G2 Preparation of plasmid DNA

25 Grow the cell Harvest the cell by centrifugation Alkaline lysis of the cell Resuspend the cell pellet neutralization Phenol extraction Ethanol precipitation CsCl gradient purification

26 G2- 6 Cesium chloride gradient centrifugation 1.CsCl gradient purification is the last step of large scale plasmid DNA purification 2.Laborious 3.Best for the production of very pure supercoiled plasmid DNA 4.The presence of ethidium bromide (EB) is important. Binding of EB to DNA will unwind the DNA and reduce the DNA density Supercoilded DNA bind less EB than linear DNA or nicked DNA, thus has a higher density Supercoiled DNA may be purified from protein,RNA chromosomal DNA and nicked plasmid DNA in one step!! G2 Preparation of plasmid DNA

27 G3 Restriction Enzymes and electrophoresis G3-1 Restriction endonuclease G3-2 Recognition sequences G3-3 Cohesive ends G3-4 Restriction digests G3-5 Agarose gel electrophoresis G3-6 Isolation of fragments Gene manipulation

28 G3-1 Restriction endonuclease 1.Bacterial enzymes which cut DNA into defined and reproducible fragments 2.Identified in the 1960s 3.Key discovery which allowed the DNA cloning to become a reality G3 Restriction enzymes and electrophoresis

29 One component of the bacterial restriction- modification system, a natural defense mechanism of bacteria to against the introduction of foreign DNA into the cell Restriction 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 G3 Restriction enzymes and electrophoresis

30 G3-2&3 Restriction sequences&Cohesive ends 5’ protruding ends 3’ protruding ends 5’-CCCGGG-3’ 3’-GGGCCC-5’ 5’-CCC-OH 3’-GGG- p p -GGG-3’ OH-CCC-5’ + SmaI blunting ends Cohensive ends G3 Restriction enzymes and electrophoresis

31 Recognition sequences Restriction enzymes 1.Recognize 4-8 bp. Most recognition sequences are 6 bp which occurs at a rate of 4 6 =4096 bp. 2.Highly specific 1.Commercially available 2.Require Mg 2+ for enzymatic activity G3 Restriction enzymes and electrophoresis

32 G3-4 Restriction digestion G3 Restriction enzymes and electrophoresis

33 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

34 Agrose gel electrophoresis G3 Restriction enzymes and electrophoresis

35 Isolation of fragments and Agarose gel electrophoresis insert 1.Restriction digestion 2.Agarose gel electrophoresis 3.Gel excision and purification 4.Ligation with vector 5.transformation G3 Restriction enzymes and electrophoresis

36 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

37 G4-1 Alkaline phophatse removes 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 ligation Single restriction enzyme directed cloning G4 Ligation, transformation and analysis of recombinants

38 DNA ligation Covalently join the DNA molecules with the base-pairing cohesive ends, or blunt ends, if the 5’-ends have phosphate groups. G4-2&3 DNA ligation & recombinant DNA molecules G4 Ligation, transformation and analysis of recombinants

39 X if the vector is phosphoralated Recombinant DNA molecules G4 Ligation, transformation and analysis of recombinants

40 The use of alkaline phosphate to prevent religation of vector molecules G4 Ligation, transformation and analysis of recombinants

41 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

42 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

43 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

44 G4-7 Growth and storage of transformants 1.Can be grow in liquid broth or solid plates 2.Maintain the selection pressure by the presence of the corresponding antibiotics. Plasmid stability and lose 3.Store the transformant bacteria by freezing a portion in the presence of glycerol to protect from ice crystal formation G4 Ligation, transformation and analysis of recombinants

45 G4-8&9 Gel analysis and fragment orientation 1.Distinguish the recombinant plasmids from the recreated vectors by Size of the plasmids (not work well if the plasmids are prepared with alkaline lysis) Restriction digestion 2. 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 G4 Ligation, transformation and analysis of recombinants

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47 3.Analysis of a clone by restriction mapping G4 Ligation, transformation and analysis of recombinants

48 Thanks


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