- Genetic engineering involves the manipulation of naturally occurring processes and enzymes.
- extraction/syntheses a gene(s) from one organism
- transfer gene(s) into another organism (of same or different species)
- gene is expressed in new host
Stages involved
- Obtain a copy of the required gene
- place the gene in a vector
- introducing the vector DNA into the host cell
- select the cells which have taken up the foreign DNA
Plasmids as vectors in gene cloning
- Plasmids are small, circular pieces of double-stranded DNA
- small ie they are easy to use
- exist naturally in bacteria ie bacteria take up plasmids from surroundings
- can be produced artificially
- double stranded: can insert genes from prokaryotes and eukaryotes
- replicate independently in bacteria
- can be transferred between different bacterial species
Enzymes used
- Restriction endonuclease:
- restrict viral infections by recognizing and breaking down DNA of invading viruses.
- binds to specific target site on DNA (sequence of bases) and cut sugar phosphate backbone
- straight across produce blunt ends
- staggered fashion produce sticky ends
- sticky ends: short lengths of unpaired bases; easily form hydrogen bonds with complementary base sequences on other fragments of DNA cut with the same restriction enzyme
- Reverse transcriptase:
- uses single-stranded mRNA as the template
- reverses the transcription process: produces single stranded DNA
- single-stranded DNA + DNA polymerase = double-stranded complementary DNA (cDNA)
DNA ligase links together sugar-phosphate backbones of DNA molecules and plasmid produces a closed circle of double-stranded DNA containing the new gene ie recombinant DNA