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Gene cloning

gene cloning basics...

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Gene cloning

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