Cloning and genetic engineering by Ivo Frébort

Cloning Clone: a collection of molecules or cells, all identical to an original molecule or cell To "clone a gene" is to make many copies of it - for example, in a population of bacteria To "clone a gene" is to make many copies of it - for example, in a population of bacteria Gene can be an exact copy of a natural gene Gene can be an exact copy of a natural gene Gene can be an altered version of a natural gene Gene can be an altered version of a natural gene Recombinant DNA technology makes it possible Recombinant DNA technology makes it possible

Plasmids Naturally occurring extrachromosomal DNA Plasmids are circular dsDNA Plasmids are circular dsDNA Plasmids can be cleaved by restriction enzymes, leaving sticky ends Plasmids can be cleaved by restriction enzymes, leaving sticky ends Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid

Cloning Vectors Plasmids that can be modified to carry new genes Plasmids useful as cloning vectors must have Plasmids useful as cloning vectors must have –a replicator (origin of replication) –a selectable marker (antibiotic resistance gene) –a cloning site (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers

DNA Libraries DNA Libraries Sets of cloned DNA fragments that together represent the genes of a particular organism Any particular gene may represent a tiny, tiny fraction of the DNA in a given cell Any particular gene may represent a tiny, tiny fraction of the DNA in a given cell Can't isolate it directly Can't isolate it directly Trick is to find the fragment or fragments in the library that contain the desired gene Trick is to find the fragment or fragments in the library that contain the desired gene

Colony Hybridization Disk is treated with base or heated to convert dsDNA to ssDNA and incubated with probes Disk is treated with base or heated to convert dsDNA to ssDNA and incubated with probes Colonies that bind probe hold the fragment of interest Colonies that bind probe hold the fragment of interest

Colony Hybridization

Southern Blots

The Polymerase Chain Reaction What if you don't have enough DNA for colony hybridization or Southern blots? The small sample of DNA serves as template for DNA polymerase The small sample of DNA serves as template for DNA polymerase Make complementary primers Make complementary primers Add primers in more than 1000-fold excess Add primers in more than 1000-fold excess Heat to make ssDNA, then cool Heat to make ssDNA, then cool Run DNA polymerase (usually Taq) Run DNA polymerase (usually Taq) Repeat heating, cooling, polymerase cycle Repeat heating, cooling, polymerase cycle

DNA sequencing Chemical Cleavage Method Four reactions are used G-specific cleavage with dimethyl sulfate, followed by strand scission with piperidine G-specific cleavage with dimethyl sulfate, followed by strand scission with piperidine G/A cleavage: depurination with mild acid, followed by piperidine G/A cleavage: depurination with mild acid, followed by piperidine C/T cleavage: ring hydrolysis by hydrazine, followed by piperidine C/T cleavage: ring hydrolysis by hydrazine, followed by piperidine C cleavage: same method (hydrazine and piperidine), but high salt protects T residues C cleavage: same method (hydrazine and piperidine), but high salt protects T residues

DNA sequencing Chain Termination Method Based on DNA polymerase reaction Run four separate reactions Run four separate reactions Each reaction mixture contains dATP, dGTP, dCTP and dTTP, one of which is P-32-labelled Each reaction mixture contains dATP, dGTP, dCTP and dTTP, one of which is P-32-labelled Each reaction also contains a small amount of one dideoxynucleotide: either ddATP, ddGTP, ddCTP or ddTTP Each reaction also contains a small amount of one dideoxynucleotide: either ddATP, ddGTP, ddCTP or ddTTP

Recombinant Proteins

Plant transformation Common Strategies for Introducting Foreign Genes into Plant Cells: Agrobacterium infection Agrobacterium infection Particle bombardment Particle bombardment Electroporation Electroporationhttp://

Regeneration of transgenic plant from crown-gall callus

Tobacco plants overexpressing HvCKX genes

Transgenic Animals Genes can be introduced into animals by transfection - injection of plasmid DNA into recipient cells Genes can be introduced into animals by transfection - injection of plasmid DNA into recipient cells Plasmids can be injected into fertilized eggs in mice Plasmids can be injected into fertilized eggs in mice Expression is usually variable, because the gene is inserted randomly Expression is usually variable, because the gene is inserted randomly Growth hormone transfection produces mice that are very large! Growth hormone transfection produces mice that are very large!