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  • 1. GENE CLONING
    BHUPENDRA SINGH RATHORE
    B.sc. Biotech (II Sem.)
  • 2. GENE CLONING
    What does the term cloning mean?
    What is gene cloning? How does it differ from cloning an entire organism?
    Why is gene cloning done?
    How is gene cloning accomplished ?
    What are some of the ethical considerations regarding gene cloning?
  • 3. CLONING-A DEFINITION
    From the Greek - klon, a twig
    An aggregate of the asexually produced progeny of an individual; a group of replicas of all or part of a macromolecule (such as DNA or an antibody)
    An individual grown from a single somatic cell of its parent & genetically identical to it
  • 4. What is DNA cloning ?
    When DNA is extracted from an organism, all its genes are obtained
    In gene (DNA) cloning a particular gene is copied (cloned)
  • 5. What is gene cloning ?
    • To "clone a gene" is to
    make many copies of it
    • Act of making many
    identical copies of gene
    • Gene can be an exact
    copy of a natural gene
    • Gene can be an altered
    version of a natural gene
  • 6. Whole organisms are cloned too, but differently
  • 7. Why we 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.
  • 8. HOW IS DNA CLONED ?
    Cell based DNA cloning
    Cell-free DNA cloning (PCR)
  • 9. Cell based DNA cloning
    Blood sample
    DNA is extracted- here from blood
    Restriction enzymes, e.g. EcoRI, HindIII, etc., cut the DNA into small pieces
    Different DNA pieces cut with the same enzyme can join, or recombine.
    DNA
    RESTRICTION ENDONUCLEASE
  • 10. DNA CLONING I
    The action of a restriction enzyme, EcoRI
    Note: EcoRI gives a ‘sticky’ end
  • 11. DNA CLONING II
    • Bacterial plasmids (small circular DNA additional to a bacteria’s regular DNA) are cut with the same restriction enzyme
    • 12. A chunk of DNA can thus be inserted into the plasmid DNA to form a “recombinant”
  • DNA CLONING III
    • The recombinant plasmids are then mixed with bacteria which have been treated to make them “competent”, or capable of taking in the plasmids
    • 13. This insertion is called transformation
  • DNA CLONING IV
    The plasmids have naturally occurring genes for antibiotic resistance.
    Bacteria containing plasmids with these genes will grow on a medium containing the antibiotic- the others die, so only transformed bacteria survive
  • 14. Antibiotic resistance(screening)
    The medium in this petri dish contains the antibiotic Kanamycin.
    The bacteria on the right contain Kanr, a plasmid that is resistant to Kanamycin, while the one on the left has no resistance.
    Note the difference in growth.
  • 15. DNA CLONING V
    • The transformed bacterial cells form colonies on the medium
    • 16. Each cell in a given colony has the same plasmid (& the same DNA)
    • 17. Cells in different colonies have different plasmids (& different DNA fragments)
  • Cell free DNA cloning (PCR)
    Developed in the mid 1980s.
    Kary Mullis - Nobel prize (1993).
    Revolutionized molecular biology.
    DNA fragments can be amplified in large amounts.
    In vitro technique.
  • 18. What do we need for PCR?
    Sequence information
    Oligonucleotide primers
    DNA
    Nucleotides
    Heat-stable DNA polymerase
    Taq(Thermusaquaticus)
  • 19. PCR technique
    ds DNA
    Step 1
    Denature
    Step 2
    Anneal
    Step 3
    Extend
  • 20. PCR amplifies DNA(gene)
    1st cycle
    1st cycle
    2nd cycle
    2nd cycle
    3rd cycle
    4th cycle
    5th cycle
    6th cycle
  • 21. Any questions
  • 22. THANKSSS !!!