Cloning
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Cloning

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Cloning Cloning Presentation Transcript

  • RECOMBINANT DNA TECHNOLOGY
  • What is Genetic Engineering?
    • Genetic engineering (GE) is the transfer of genes from one organism to another through means that do not occur in nature, but through human intervention. This involves isolating and then moving genes within and without different species by recombinant DNA techniques and other manipulation of the genetic construct outside the traditional practices such as sexual and asexual breeding, hybridization, fermentation, in-vitro fertilization and tissue culture.
  • The Term of Gene Manipulation
    • Gene Manipulation is defined as the formation of new combinations of heritable material by the insertion of nucleic acid molecules , produced by whatever means outside the cell, into any virus, bacterial plasmid or other vector system so as to allow their incorporation into a host organism in which they do not naturally occur but in which they are capable of continued propagation. Also named gene cloning .
    View slide
    • Gene cloning is production of many identical copies of the same gene.
      • If the inserted gene is replicated and expressed, we can recover the cloned gene or protein product.
      • Cloned genes have many research purposes: determining the base sequence between normal and mutated genes, altering the phenotype, obtaining the protein coded by a specific gene, etc.
      • Humans can be treated with gene therapy : alteration of the phenotype in a beneficial way
    DNA Manipulation View slide
    • Recombinant DNA (rDNA) contains DNA from two or more different sources
      • Requires:
        • A vector
          • introduces rDNA into host cell
          • Plasmids (small accessory rings of DNA from bacteria) are common vectors
          • Phage vectors (bacterial viruses) can also be used
        • Two enzymes to introduce foreign DNA into vector DNA
          • A restriction enzyme - cleaves DNA
            • Bacterial enzyme that stops viral reproduction by cleaving viral DNA
            • Act as molecular scisssors (cut plasmids and foreign human DNA)
            • Produce short single stranded “sticky ends” where insertions of foreign DNA can be made
          • A DNA ligase enzyme - seals DNA into an opening created by the restriction enzyme
    DNA Manipulation
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  • Cloning a Human Gene Restriction enzyme Eco RI
            • Bacterial enzyme that stops viral reproduction by cleaving viral DNA
            • Act as molecular scisssors (cut plasmids and foreign human DNA)
            • Produce short single stranded “sticky ends” where insertions of foreign DNA can be made
  • What is a Gene? a Genome?
    • A gene is a unit of inheritance. For example, children tend to look like their parents. We inherit our features through our genes, half of which come from one parent, and half from the other.
    • A gene is also defined by a digital code of just four DNA bases (A,T,G & C) that is nearly universal for all known life forms, whether viral, bacterial, fungal, plant, animal or human. The average size gene of bacteria is about 1,000 bases long.
    • Since genes are encoded by the DNA bases that comprise the linear strands of a chromos-ome, the genes are arranged in linear order along chrom-osomes, and they can be mapped.
  • Why Clone DNA?
    • A particular gene can be isolated and its nucleotide sequence determined
    • Control sequences of DNA can be identified & analyzed
    • Protein/enzyme/RNA function can be investigated
    • Mutations can be identified, e.g. gene defects related to specific diseases
    • Organisms can be ‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc.
  • How is DNA cloned? Cell-based DNA cloning Cell-free DNA cloning (PCR)
  • Clone in dividing cells
  • Clone in P C R
  • Restriction Endonucleases --The Molecular Scissors Host enzymes that prevent the invasion of foreign DNAs such as viral DNA, by cutting them up. These enzymes cut within the foreign DNAs, rather than chewing them away from the ends. These enzymes recognize a specific DNA sequence (4-12bp) which is twofold symmetry and cut both DNA strands Some enzymes make staggered cuts GAATTC CTTAAG Restriction Endonucleases Some make even cuts CCCGGG GGGCCC
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  • Sticky end Sticky end
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  • DNA ligase covalently links two DNA strands Restriction enzyme Restriction enzyme Ligase Ligase 5’ 3’ 5’ 3’
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  • Plasmid: a cloning vector or vehicle
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    • Restriction Enzyme Mechanisms:
    • Preparation of DNAs to be joined
    • (a)Staggered cut: leaves “sticky ends”
    • Restriction Enzyme Mechanisms: Preparation of DNAs to be joined:
    • (b) Blunt End
  • Ligation of DNA cut with a Restriction Enzyme
    • Staggered “sticky ends”
  • Ligation of DNA cut with a Restriction Enzyme
    • Role of T4 DNA Ligase
  • Selectable Markers:
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  • cDNA Library
    • Used to obtain functional eukaryotic coding regions.
    • E. coli does not process introns.
    • First step: Isolate poly A+ mRNA with oligo (dT) cellulose
  • cDNA Library preparation
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  • Cloning vectors
    • Cloning vectors are carrier DNA molecules. Four important features of all cloning vectors are that they:
    • (i) can independently replicate themselves and the foreign DNA segments they carry;
    • (ii) contain a number of unique restriction endonuclease cleavage sites that are present only once in the vector;
    • (iii) carry a selectable marker (usually in the form of antibiotic resistance genes or genes for enzymes missing in the host cell) to distinguish host cells that carry vectors from host cells that do not contain a vector; and
    • (iv) are relatively easy to recover from the host cell.
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  • Plasmids
    • Naturally occurring extrachromosomal DNA
    • Plasmids are circular dsDNA
    • Plasmids can be cleaved by restriction enzymes, leaving sticky ends
    • Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid
  • Vectors -- the DNA carriers Must have a origin of replication Allow the vector as well as the foreign DNA to amplify in the host cell
    • Plasmids
    2) Phages Allow the host to grow on selective media Can selectively amplify this specific vector in the host cell Allow insertion of foreign DNA Origin of replication Antibiotic-resistant genes Multiple cloning sites
  • Plasmids
    • Recombinant DNA vectors:
      • Amplification of DNA fragment can be achieved in the cell using cloning vectors: plasmid or bacteriophages
      • Plasmid
    Small circular DNA in bacteria or yeast cells Accumulate 1-5 kb inserts LacZ encodes  -galactosidase Lacl – encodes factor controling transcription of lacZ
  • Bacteriophage λ (Lambda)
    • For cloning inserts of 10-20 Kb
    • Plasmid libraries hold up to 10 kb inserts
  • Bacteriophage λ (Lambda)Life Cycle
    • Lytic Cycle:Production of progeny
    • Lysogenic Cycle: Integration into bacterial chromosome
  • BACs: Bacterial Artificial Chromosomes
    • Based on P1 bacteriophage, the F plasmid and the lacZ region of pUC plasmids
    • It’s a low copy number plasmid
    • Carries 50-300kb fragments
  • BACs: Bacterial Artificial Chromosomes
  • YACs:Yeast Artificial Chromosomes
    • Transformation
    • Electroporation
    • Protoplast fusion
    DNA can be inserted into a cell by:
    • Microinjection
    • Gene gun
    DNA can be inserted into a cell by:
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  • Bacterial transformation Introduction of DNA into bacteria Spontaneous uptake – low probability E. coli – cells treated with CaCl 2 Less than 1 of 10 3 cells acquire a plasmid Selection of transformed cells: resistance to antibiotics using chromogenic substances Antibiotics: molecules produced by microorganism that kill other microorganism peniciline, tetracycline, ciplroflaxine – inhibits gyrase in the complex with DNA – inhibits DNA replication Chromogenic substances:
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  • Screening
    • The medium in this petri dish contains the antibiotic Kanamycin
    • The bacteria on the right contain Kan r , a plasmid that is resistant to Kanamycin, while the one on the left has no resistance
    • Note the difference in growth
  • Propagation
    • Once colonies are identified, they are cultured in broth to increase numbers and therefore the amount of DNA
    • Samples are also prepared for storage at -80 degrees. They can be kept for many years this way.
  • Agricultural Applications
    • Herbicide resistance
    • -Broadleaf plants have been engineered to be resistant to the herbicide glyphosate
  • Agricultural Applications
    • Pest resistance
    • -Insecticidal proteins have been transferred into crop plants to make them pest-resistant
    • - Bt toxin from Bacillus thuringiensis
    • Golden rice
    • -Rice that has been genetically modified to produce  -carotene (provitamin A)
    • -Converted in the body to vitamin A
  • Agricultural Applications
    • Adoption of genetically modified (GM) crops has been resisted in some areas because of questions about:
    • -Crop safety for human consumption
    • -Movement of genes into wild relatives
    • -Loss of biodiversity
  • Agricultural Applications
    • Biopharming
    • -Transgenic plants are used to produce pharmaceuticals
    • -Human serum albumin
    • -Recombinant subunit vaccines
    • -Against Norwalk and rabies viruses
    • -Recombinant monoclonal antibodies
    • -Against tooth decay-causing bacteria
  • Transgenic Mammals
    • The insertion of genetic material into human cells for the treatment of a disorder
    Medical Applications
  • Recombinant DNA Vaccines? Strategy for a subunit vaccine for herpes simplex
  • Gene Therapy Treatment of SCID (severe combined immunodeficiency). SCID affects the maturation of immune cells that develop in bone marrow. SCID sufferers lack the enzyme ADA (adenosine deaminase).