Recombinant DNA technology - in vivo amplification Flashcards
During in vivo amplification, describe how a gene could be inserted into a plasmid.
Cut the plasmid using the same restriction endonuclease as the fragment; So that both have complementary sticky ends; Mix together the fragments and cut plasmids and add the enzyme DNA ligase to join the complementary sticky ends;
- Explain why it is important to use the same restriction enzyme on both pieces of DNA.
To cut the DNA at the same recognition sequence; so the sticky ends are complementary;
Explain how sticky ends are useful in genetic engineering.
To cut the DNA at the same recognition sequence; so the sticky ends are complementary;
Joining two pieces of DNA; By complementary base-pairing;
Why do DNA fragments need a promoter and terminator regions adding to them?
A fragment created using reverse transcriptase and mRNA has no promoter and terminator regions. The promoter and terminator regions tell RNA polymerase where to start and finish transcription, so they need to be incorporated into the fragment that is being inserted into an organism to produce a specific protein.
What is the role of a vector?
Vectors are used to transport the DNA fragment into the host cell.
What are the roles of enzymes in the insertion of a DNA fragment into plasmid/vector?
Restriction enzymes cut the plasmid/vector; DNA ligase joins the sticky ends of the DNA fragment to the plasmid/vector via phosphodiester bonds.
Once a fragment of DNA has been inserted into a plasmid/vector. How is it amplified?
The plasmid/vector is taken up by the host cell; the host perform cell division producing many copies of the desired fragment;
What is the transformation of a host cell?
The uptake of a new DNA fragment from a different organism/species, using a vector to insert the fragment into the organisms genome. The host cell should now be able to produce a polypeptide inserted fragment codes for.
What is the role of marker genes?
Marker genes are used to detect whether the DNA fragment has been successfully inserted in to the hosts cells/to identify recombinant cells.
What is a genetically modified (GM) organism?
An organism that has had a DNA fragment from another organism/species inserted into the genome, so that it can now produce the polypeptide the DNA fragment codes for.
Explain why it is important that vectors are added to the embryos of multicellular organism (plants/animals).
If injected into embryo, gene gets into most of cells of organism;
So gets into cells that need to make the protein; As the DNA will be replicated with the host DNA inside the nucleus; Then passed to new cells during mitosis; to produce genetically identical cells;
Give examples of how marker genes can identify genetically modified (GM) organisms.
Use a plasmid with two antibiotic resistance genes; add the target gene into the middle of the first marker gene. Grow the bacteria on agar containing the antibiotic which the second marker gene gives resistance to. Create a replica plate on agar containing the antibiotic that the first resistance gene gives resistance to. Any colonies present on the first plate that did not grow on the replica plate must be recombinant.
Alternatively use a different marker gene for the second marker e.g. green fluorescent protein or an enzyme that makes a coloured product. No replica plate is needed for these as the colonies will be a different colour/fluoresce under UV light.
Explain the role of recombinant DNA technology in gene therapy.
DNA fragments containing the normal form of a gene can be added to a virus; the virus can be used to infect an individual that has a faulty version of the gene; the virus inserts the normal form of the gene into the individuals genome of target cells; the target cells can now make the normal protein for a limited period;
