state the 5 steps of DNA technology (in vivo cloning)
- create DNA fragment
- insert DNA into a vector
- transform host cell with vector
- identify transformed cell
- grow host cell (clone)
how is DNA amplified?
- using PCR
- cDNA into thermal cycler
summarise how bacteria is used to produce human insulin
- mRNA extracted from cells and treated with reverse transcriptase to make complementary DNA/plasmid obtained from bacteria & cut with restriction enzyme
- plasmid and cDNA fuse using DNA ligase
- recombinant plasmid introduced into host cells
- bacteria multiply in a fermenter and produce insulin
- separation & purification of human insulin can be used by diabetic patients
what are the advantages of using bacteria in the process?
- multiply quickly
- DNA is easily accessible (has free/plasmid DNA in cytoplasm)
describe how a plasmid is cut
- both strands of DNA cut
- using a specific restriction enzyme
- in a particular section
- creates sticky ends
how can the same sticky end be created?
use same restriction enzyme - will cut at same recognition site
why are sticky ends made to be identical?
cut plasmid & cDNA align and the cDNA becomes part of the plasmid
how can the plasmid be inserted into a bacterium?
- injected (using a vector)
- use a solvent / detergent
- increase temperature
- electric shock bacteria cell
how can bacteria that have taken up insulin be detected?
inserting an indicator / antibiotic resistant bacteria when cDNA is inserted to show those that have picked up plasmids
what is a plasmid?
a small, circular, double-stranded DNA molecule that naturally exist in bacterial cells - often provide bacteria with antibiotic resistance.
what is cDNA
copy DNA
