Recombinant DNA technology- BP Flashcards
What is genetic engineering?
changing the genetic make-up of an organism’s DNA by adding or removing a gene
the DNA becomes Recombinant
the Organism becomes Genetically Modified (Transgenic)
why do we genetically engineer animals?
to give them additional characteristics
so they can make useful products (proteins)
Examples of genetic engineering in animals?
additional characteristics:
add gene for disease resistance
add gene for growth hormone for growth
making useful products:
use to produce anti-thrombin = protein used to make blood clot (people with certain genetic disease may not produce), use milk producing animal to produce, add gene for anti-thrombin next to milk producing gene in animal, therefore anti-thrombin protein will be made in the milk (easily extracted)
why do we genetically engineer plants?
to give them additional characteristics
so they can make useful products (proteins)
Examples of genetic engineering in plants?
Additional characteristics:
add gene for disease resistance
add gene for pest resistance
add gene for pesticide resistance
add gene to promote growth for high yield
produce genetically modified tomatoes = prevented from softening therefore remain hardened (easy for storage and transport), involves preventing formation of softening enzyme, a gene is added that is complementary to the the softening enzyme gene, so its mRNA will bind to the mRNA of the softening enzyme preventing translation of the softening enzyme Making useful products:
use to make golden rice (rice that contains beta-carotene, a pre-cursor to vitamin A to treat malnutrition deficiency)
use to make protein raw material for polymers
why do we genetically engineer bacteria?
so they can make useful products (proteins)
Genetically engineering bacteria?
to make useful proteins e,g, Insulin
used to use animal sources (problems = limited supply, infection risk, immunorejection)
involves adding human insulin gene to a plasmid, then inserting this into a bacteria = the bacteria now has the gene/code to produce the human insulin protein
involves 5 steps =
1. Isolation
2. Insertion
3. Transformation
4. Identification
5. Growth/Cloning
what is isolation in terms of genetically engineering bacteria?
either by Reverse Transcriptase or Restriction Endonuclease or Gene Machine
what is insertion in terms of genetically engineering bacteria?
cut plasmid using the same RE from isolation stage
leaves complementary sticky ends
join human insulin gene with plasmid via the sticky ends
use DNA Ligase to join the sugar-phosphate backbone
= Recombinant plasmid (carrying human insulin gene)
what is transformation in terms of genetically engineering bacteria?
mix recombinant plasmid with bacteria
add Calcium ions and heat shock
bacteria will become permeable and take up the recombinant plasmid
= Genetically Modified Bacteria (carrying recombinant plasmid with human insulin gene)
what is identification in terms of genetically engineering bacteria?
identify which of the bacteria have taken up the recombinant plasmid and of these which ones have accepted the new gene (human insulin gene)
steps of identification in terms of genetically engineering bacteria?
step 1:
- choose a plasmid that carries an Ampicillin Resistance Gene
- means that when Ampicillin is added only the bacteria that have taken up the recombinant plasmid will survive (as they will have obtained the ampicillin resistance gene)
step 2:
- use gene markers (antibiotic resistant, fluorescent, enzyme) to identify which of the remaining bacteria have accepted the human insulin gene
- the human insulin gene will be placed in the middle of these gene markers
- if the bacteria accepts the human insulin gene they will reject the gene marker & if the bacteria rejects the human insulin gene they will accept the gene marker
antibiotic resistant = tetracycline resistance gene lost if human insulin gene accepted, so bacteria no longer resistant to tetracycline, add tetracycline by replica plating (on another plate that carries a few of the bacteria from each colony in their same position), the ones that die are the ones that we want, identify on original plate
fluorescent = fluorescent gene lost if human insulin gene accepted, so identify bacteria showing no fluorescence
enzyme = enzyme gene lost if human insulin gene accepted, therefore add colourless substrate, where there is no colour change select those bacteria (as enzyme not made to breakdown colourless substrate for colour change)
end result = Genetically Modified Bacteria
what is growth/cloning in terms of genetically engineering bacteria?
grow genetically modified bacteria (carrying human insulin gene)
they will produce the protein (human insulin)
What is PCR?
- polymerase chain reaction
- used to replicate DNA artificially
Polymerase Chain Reaction vs Semi-Conservative Replication?
PCR can only replicate short DNA fragments, SCR can replicate whole DNA
PCR use 95°C , SCR uses DNA Helicase
PCR uses primers, SCR does not require primers
