16.1 Producing DNA Fragments
Diabetes - insulin is a protein product of a gene - gene faulty insulin not produced - injecting it is costly + risks rejection/infection.
Can now extract clone and transfer genes into microorganisms act as factory for production of insulin and other proteins - DNA from 2 different orgs = recombinant DNA forming a genetically modified organism.
Process - isolate gene - insert gene into vector - transfer in to host cell - use gene marker to identfy host cells - cloning/growth of pop. of cells.
Insulin example - Bcells from islets of langerhans- mRNA extracted - reverse transcriptase converts mRNA to complimentary DNA - DNA polymerase builds up complementary nucleotides on cDNA = required gene.
Restriction endonucleases cut up DNA at a specific base sequence called the recognition sequence - Blunt end - straight cut through opposite base pairs - Sticky end staggered cut leaving unpaired bases on each end , sequence of unpaired bases is a palindrome.
16.2 In Vivo
In vivo is in living tissue - DNA is cut at restriction site if cut is staggered = unpaired nucleotide bases = sticky , any DNA cut by same endonuclease are complimentary and can be paired up and joined up by DNA ligase.
After they have been cut insert them into a vector , usually a plasmid with two different antibiotic resistance genes - Endonuclease cuts the plasmid so that the desired gene will fit into it (complementary) and will prevent the antibiotic resistance gene that it has been inserted into from working.
Mix plasmids with bacterial cells from the host in a medium containing calcium ions that increase the permeability of the membranes meaning up take of the plasmids is easier.
You will need to identify which of the cells contain the gene as some plasmids will not have encorporated the gene fragment and so bacteria will not have taken up the plasmids, to do this : use gene markers :
Antibiotic resistance - add the bacterial cells to a medium that contains the anitbiotic that the gene is no longer resistant to , then tranfer them to a plate containing the antibiotic that they are still resistant to, those that die on the first plate but survive on the second are the cells that contain the gene.
Fluorescent markers - uses gene from jelly fish that produces green fluorescent protein, the gene to be cloned in then placed into the centre of the jelly fish gene, the bacteria that take up the plasmid will fluoresce, this is faster than replica plating.
Enzyme markers- use gene that produces lactase (this turns a particular colourless substrate blue) , the required gene is then spliced into the lactate gene , when bacteria are grown in a colourless substrate they will be unable to change colour, gener is damaged.
16.3 In Vitro
Polymerase chain reaction is a method of copying DNA fragments , automated process , very fast. Doesnt require living cells, no risk of contamination…