Dolly the sheep (1997-2003)

1. 2. Gene cloning Cloning 1. Animal and human cloning Dolly the sheep (1997-2003)

2. 1. Animal and human cloning

3. What is Gene cloning? To "clone a gene" is to make multiple copies of it in vivo

4. Why Clone DNA? • A particular gene can be isolated and its nucleotide sequence determined • 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

5. Ligation RE1 RE1 RE2 RE2 Insulin overexpression in bacteria Gene encoding insulin RE1 RE2 RE1 RE2

6. Transformation into E. coli cells Insulin Insulin overexpression in bacteria

7. Target DNA What do we need for gene cloning? • Insert • Plasmid Vector • Ligation of the palsmid+Insert • transformation Plasmid vector ligation transformation

8. Plasmids are Used to Replicate a Recombinant DNA Plasmids are small circles of DNA found in bacteria. Plasmids replicate independently of the bacterial chromosome. Pieces of foreign DNA can be added within a plasmid to create a recombinant plasmid.

9. Plasmid vectors A Plasmid (vector) is a DNA molecule used for transferring foreign DNA fragments (genes) into host cells Circular DNAmolecules capable of autonomous replication in living cells All plasmids contain: 1. Origin of replication (ori) 2. Selection marker (Ampicillin) 3. Multiple Cloning Site (MCS) MCS

10. RE1 RE1 RE2 RE1 RE2 RE2 Insert – Target DNA 1. PCR product T T 2. Restriction Enzymes

11. REs will produce ends that enable the gene to be spliced into a plasmid Ligation Ligation

12. 1 1 1 1 DNA ligase REs and DNA ligase Ligation of the insert to the plasmidcut with only one enzyme

13. Ligation of the insert to the plasmidcut with only two enzyme RE1 RE2 ligation There is two possible outcomes

14. Transformation Use of bacterial cells to amplify the DNA of interest • Two main methods: • Chemical transformation – Chilling cells in the presence of Ca2+ prepares the cell walls to become permeable to plasmid DNA. Cells are briefly heat shocked which causes the DNA to enter the cell • Electoporation- making holes in bacterial cells, by briefly shocking them with an electric field of 10-20kV/cm. Plasmid DNA can enter the cell through these holes.

15. Possible products of the transformation: No plasmid No ampicillin resistance Plasmid without insert Ampicillin resistant Plasmid + insert Ampicillin resistant Plasmid + insert Ampicillin resistant How can we differentiate between the bacteria containing plasmid+insert and the ones with the self ligated plasmid (no insert)?

16. Our lab experimemet Insert that was amplified by PCR pGEM Vector ligation Transformation Screening

17. Possible products of the transformation: No plasmid No ampicillin resistance Plasmid without insert Ampicillin resistant Plasmid + insert Ampicillin resistant Plasmid + insert Ampicillin resistant How can we differentiate between the bacteria containing plasmid+insert and the ones with the self ligated plasmid (no insert)?

18. Cloning procedure transformation + IPTG + X-Gal

19. RNA pol. promotor LacZ gene LacZ mRNA Ribosome Lac Z gene Gene expression dogma DNA RNA β-galactosidase Protein X-gal BLUE colonies X-gal WHITE colonies

20. promotor LacZ RNA pol. IPTG IPTG IPTG IPTG IPTG IPTG promotor promotor operator operator LacZ LacZ RNA pol. RNA pol. Repressor Lac Z gene Β-galactosidase X-gal X + galactose Cells which produce ß-galactosidase form BLUE colonies. Cells without ß-galactosidase production form WHITE colonies.

21. X X X X X without plasmid Plasmid without Insert Plasmid +Insert Screening Insert T LacZ promotor operator T pGEM WHITE colonies BLUE colonies

22. Confirmation by digestion with restriction enzyme and separation of the digestion products on agarose gel A plasmid DNA will be purified from the bacteria cells. Plasmid DNA will be digested with EcoRI, and analyzed by gel electrophoresis for identification of the clone containing insert. EcoRI Vector pGEM EcoRI Insert