Genetic engineering Flashcards
transgenic/GMO
an organism that carries a gene from another organism
sticky ends
regions of unpaired exposed bases
most common technique to isolate a desirable gene?
use restriction endonucleases (enzymes) to cut the required gene frm the DNA of an organism at recognition site
- each endonuclease is restricted the breaking the DNA strands at specific base sequences within the molecule
- some make blunt cuts across double strand however most cut two strands unevenly, creating sticky ends, which make it easier to insert desired gene
less common method of isolating desired gene
isolate mRNA for desired gene and use reverse transcriptase enzyme to produce a single strand of complementary DNA
+ makes it easier to identify desired gene, as only particular cells make particular mRNA eg. B pancreatic cells make insulin, so make lots of insulin mRNA
3 stages of genetically engineering something
isolate desired gene
form recombinant DNA
transfer the vector
why are plasmids good vectors?
- separate from chromosomal DNA so can replicate independently
- contain marker gene for antibiotic resistance so scientists can tell if bacteria has taken up a plasmid by growing the bacteria in media containing the antibiotic
recombinant DNA
plasmid or other vector combined with host dna
how to insert a DNA fragment into a plasmid
- use same restriction endonucleases used to isolate desirable gene to cut plasmid, so plasmid has complementary sticky ends to DNA fragment
- DNA ligase enzyme forms phosphodiester bonds between nucleotides on backbone
- plasmid given 2nd marker gene to show the plasmid contains the recombinant DNA (usually flurescence). if the bacteria does NOT fluoresce it has been engineered successfully and can grow on
transferring the vector
- transformation
- culture bacteria cells and plasmids in calcium rich sol and increase temp, causing bacterial membrane to increase in permeability and plasmids can enter
OR - electroporation
- apply small electrical current to bacteria, making membranes v porous so plasmids can move into cells
-must carefully control electric current as membrane can be permanently damaged or destroyed, which destroys whole cell
how can electroporation be used in eukaryotic cells?
- can also be used to get DNA fragments into eukaryotic cells (DNA passes through cell membrane and nuclear membrane to fuse with nuclear DNA)
electrofusion in plant
- removal of plant cell wall by cellulases
- tiny electric currents applied to the membranes of two different cells
- this fuses the cell and nuclear membranes of the cells together to form a hybrid/polyploid cell, containing DNA from both
- plant hormones used to stimulate growth of new cell wall
- callus formation
electrofusion in animals
- do not fuse as easily and effectively as plant cells
- polyploid animal cells do not usually survive in an organism
- used in production of monoclonal antibodies
monoclonal antibody
- combination of cell producing one type of antibody and a tumour cell (which divides rapidly in culture)
- used to identify pathogens in animals and plants and in the treatment of some diseases incl. cancer
how to genetically engineer plants
- agrobacterium tumefaciens (causes tumours in healthy plants)
- desired gene placed in the Ti plasmid of A.tumefaciens along w marker gene, this is carried directly into the plant cell DNA
- transgenic plant cells form a callus
callus
mass of GM plant cells, each of which can be grown into a new transgenic plant
