Genetic Engineering

• copy of a specific gene • cloning a gene means isolating an exact copy of a single gene from the genome and copying it into a smaller more accessible factor (like a plasmid)

• obtain a pure sample of a gene and determine its nucleotide sequence • by expressing this specific DNA/protein the protein’s function can be investigated • mutations can also be identified • ‘engineer’ organisms for our use

• DNA purified from a cell. fragment of DNA containing a gene of interest is isolated using a restriction enzyme or PCR • DNA fragment is inserted into a circular DNA molecule, vector, like a plasmid to make a recombinant DNA molecule • transform host cells with the vector which then replicates producing numerous identical copies of itself and the genes it carries • host cell divides to form copies of recombinant DNA passed to progeny: further vector replication

Isolate DNA from organism with the gene of interest: • lyse cell with physical or chemical methods (sonication or homogenisation) • remove membrane lipids with detergent • remove proteins with protease • remove RNA with RNase • precipitate DNA with alcohol

• use endonucleases to fragment DNA

• origin of replication: DNA sequence ensuring the plasmid is replicated during the cell cycle • antibiotic resistance gene • multiple cloning site: series of restriction enzyme sites used to connect the fragment/gene to the plasmid

• non directional (can’t tell which way the gene will be oriented) • self ligation of the vector can occur (plasmid reattaches to itself rather than the insert- this is more likely). the vector needs to be dephosphorylated to minimise this • alkaline phosphatases used to remove phosphate from the plasmid

• directional cloning • no dephosphorylation needed • two endonucleases used to cleave plasmid leaving two complementary stick ends for the insert

• no hybridization of ends • non directional • need a phosphate treatment • is much slower than stick end ligation

• cutting out target bands from agarose gels • melting agarose gels at around 50 degrees (lower than melting point of DNA) • purify DNA fragments using glass beads and silica: sodium ions attach to oxygen and attach to DNA forming a cation bridge. pure water treatment then releases the DNA

• requires information about DNA region of interest to synthesise appropriate primers • primers are oligonucleotides complementary to different regions on the 2 strands of DNA template flanking amplification region • primers hybridize to one strand of the dsDNA and one to the other strand so both primers are oriented with their 3’ ends pointing towards each other • primers are the starting point for elongation at 3’ end by the DNAP

• uses PCR based cloning • need primer with homology to DNA • end of primer contains the restriction site

• uses PCR based cloning • can prepare a complementary plasmid for insertion without endonucleases • Taq DNA polymerases add adenine to the 3’ end of the product

TOPO cloning: • Vaccinia virus topoisomerase I specifically recognises and digests DNA sequence (C/T)CCTT, and unwinds the DNA and re-ligates it again at the 3’ phosphate group of the last thymidine. • TOPO vectors carry (C/T)CCTT at the two linear ends. The linear vector DNA already has the topoisomerase enzyme covalently attached to both of its strands’ free 3’ ends. • PCR is performed to amplify target DNA fragment. • Once the PCR products and TOPO vectors are mixed, the topoisomerase catalyse ligation of the two ends at r.t. in 5 min.

Cloning by homologous recombination: • PCR is performed with primers containing overlap sequences with vectors. • DNA recombinases recognise overlapped sequences between vectors and PCR products, and catalyse the recombination and insertion of the PCR product into the vector.

• connects 3’OH to 5’P together - uses ATP for energy

• DNA introduced into cells through pores created by an electrical field

• cells become competent when incubated with CaCl2 in ice due to changes in the cell surface structure and increasing permeability to DNA • heat pulse creates thermal imbalance across membrane, allowing DNA entry through pores

• precipitation of DNA onto cell surface with Ca phosphate • introduction by liposomes • transformation of plant protoplasts i.e. plant cell after wall has been degraded • microinjection i.e. inject DNA into nucleus • biolistics i.e. transformation with microprojectiles • electroporation

• nick and displacement of nicked strand • replication by DNAP III creating ds plasmid • rejoining of nicked strand and synthesis of ds plasmid from ss circle

• Code proteins exhibit resistance to antibiotics • Act as selectable markers to identify bacteria with a particular plasmid i. e. in presence of ampicillin only cells expressing the protein for antibiotic resistance can grow • Most popular antibiotic selections: Ampicillin & Kanamycin

• irreversible inhibitor of transpeptidases • cell lysis due to inhibition of cell wall synthesis • B-lactamase breaks B-lactam ring of antibiotics so this gene confers resistance

• interacts with 30S subunit of prokaryotic ribosomes • induces substantial mistranslation + indirectly inhibits translocation during protein synthesis (cell death) • neomycin phosphotransferase II phosphorylates and inactivates aminoglycoside antibiotics like kanamycin

• used to identify recombinants • uses plasmid with modified lacZ gene coding for only part of the B-galactosidase • enzyme only synthesised when plasmid containing missing lacZ segment is present • MCS is in the middle of the lacZ gene • DNA fragment insertion into the MCS disrupts lacZ gene so it is non functional • B-galactosidase hydrolyses X-gal into a blue precipitate • therefore, recombinants are recognised by their white color