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1. Animal and human cloning Dolly the sheep (1997-2003) 2. Gene cloning 1. Animal and human cloning What is Gene cloning? To "clone a gene" is to make multiple copies of it in vivo 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 Insulin overexpression in bacteria Gene encoding insulin RE1 RE2 RE1 RE2 RE1 RE1 RE2 RE2 Ligation Insulin overexpression in bacteria Transformation into E. coli cells Insulin What do we need for gene cloning? •Insert •Plasmid Vector •Ligation of the palsmid+Insert •transformation Target DNA Plasmid vector ligation transformation 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. Plasmid vectors A Plasmid (vector) is a DNA molecule used for transferring foreign DNA fragments (genes) into host cells Circular DNA molecules 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 Insert – Target DNA 1. PCR product 2. Restriction Enzymes RE1 RE2 RE1 RE1 RE2 RE2 Ligation REs will produce ends that enable the gene to be spliced into a plasmid Ligation REs and DNA ligase Ligation of the insert to the plasmid cut with only one enzyme 1 1 1 1 DNA ligase Ligation of the insert to the plasmid cut with only two enzyme RE1 RE2 ligation There is two possible outcomes Transformation Use of bacterial cells to amplify the DNA of interest Two main methods: 1. 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 2. Electoporation- making holes in bacterial cells, by briefly shocking them with an electric field of 1020kV/cm. Plasmid DNA can enter the cell through these holes. Possible products of the transformation: Plasmid + insert Ampicillin resistant Plasmid without insert Ampicillin resistant No plasmid No ampicillin resistance How can we differentiate between the bacteria containing plasmid+insert and the ones with the self ligated plasmid (no insert)? Our lab experimemet Insert that was amplified by PCR pGEM Vector ligation Transformation Screening Possible products of the transformation: Plasmid + insert Ampicillin resistant Plasmid without insert Ampicillin resistant No plasmid No ampicillin resistance How can we differentiate between the bacteria containing plasmid+insert and the ones with the self ligated plasmid (no insert)? Cloning procedure transformation + IPTG + X-Gal Lac Z gene Gene expression dogma DNA promotor RNA pol. RNA Ribosome LacZ gene LacZ mRNA β-galactosidase Protein X-gal BLUE colonies X-gal WHITE colonies Lac Z gene promotor LacZ RNA pol. promotor operator RNA pol. LacZ Repressor IPTG IPTG promotor operator RNA pol. IPTG LacZ IPTG IPTG X-gal Β-galactosidase X + galactose Cells which produce ß-galactosidase form BLUE colonies. Cells without ß-galactosidase production form WHITE colonies. Screening Insert promotor operator T LacZ T pGEM X X X without plasmid X X WHITE colonies BLUE colonies Plasmid without Insert Plasmid +Insert 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 pGEM Vector EcoRI Insert