Gene cloning involves making many identical copies of a gene. It differs from cloning an entire organism. Gene cloning is done to isolate and study particular genes, identify control sequences and mutations, and engineer organisms for specific purposes like insulin production. It is accomplished by extracting DNA from an organism, cutting it into pieces with restriction enzymes, and inserting the gene of interest into bacterial plasmids which are then taken up by bacteria. This allows the gene to be amplified as the bacteria containing the recombinant plasmid multiply. PCR is another method that amplifies DNA fragments in vitro using heat-stable DNA polymerase and repeated heating and cooling cycles.

1. GENE CLONINGBHUPENDRA SINGH RATHOREB.sc. Biotech (II Sem.)

2. GENE CLONINGWhat 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 DEFINITIONFrom the Greek - klon, a twigAn 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 obtainedIn 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 determinedControl sequences of DNA can be identified & analyzedProtein/enzyme/RNA function can be investigatedMutations can be identified, e.g. gene defects related to specific diseasesOrganisms can be ‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc.

8. HOW IS DNA CLONED ?Cell based DNA cloningCell-free DNA cloning (PCR)

9. Cell based DNA cloningBlood sampleDNA is extracted- here from blood Restriction enzymes, e.g. EcoRI, HindIII, etc., cut the DNA into small piecesDifferent DNA pieces cut with the same enzyme can join, or recombine.DNARESTRICTION ENDONUCLEASE

10. DNA CLONING IThe action of a restriction enzyme, EcoRINote: EcoRI gives a ‘sticky’ end

11. DNA CLONING IIBacterial 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 IIIThe 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 transformationDNA CLONING IVThe 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 VThe 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 informationOligonucleotide primersDNANucleotidesHeat-stable DNA polymeraseTaq(Thermusaquaticus)

19. PCR techniqueds DNAStep 1DenatureStep 2AnnealStep 3Extend

20. PCR amplifies DNA(gene)1st cycle1st cycle2nd cycle2nd cycle3rd cycle4th cycle5th cycle6th cycle

21. Any questions