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What is recombinant DNA? DNA from two different organisms that has been
combined
What is recombinant DNA technology usually used for? To produce large quantities
of a desired protein
What are the steps involved in making a GMO for protein production? Isolation of
the gene of interest<div>Insertion of that gene into a
vector</div><div>Transformation- transfer of the vector into host
cells</div><div>Identification of the host cells that have taken up the
vector</div><div>Cloning of the host cells</div>
What are the three methods used for the isolation of the gene of interest? Using
reverse transcriptase<div>Using restriction endonucleases </div><div>Using a
gene machine, i.e. artificially synthesising the gene </div>
How can you use reverse transcriptase to isolate the gene of interest? First you
obtain the mRNA of the protein you desire<div>Then you add reverse transcriptase
which assigns DNA nucleotides to the mRNA by complementary base pairing, creating a
single stranded complementary strand of DNA</div><div>This single strand is
isolated using an enzyme</div><div>DNA polymerase is then used to create the other
strand to the DNA, creating a double stranded copy of the gene </div>
What are restriction endonucleases? Enzymes that cut DNA with a staggered cut to
leave a few exposed bases at either end on each strand. Each restriction
endonuclease is specific to a certain sequence of bases to cut
How does the gene machine work? The amino acid sequence of the desired protein
is determined, which is used in turn to find the DNA sequence of its accompanying
gene<div>The computer designs small, overlapping single strands of DNA called
oligonucleotides, which can be assembled into the gene </div><div>The
oligonucleotides are assembled, and these are assembled into the gene, which is
amplified using PCR</div><div>PCR also makes the gene double
stranded </div><div>Sticky ends are added onto this gene so it can be used in
a verctor </div>
What are the two ways of replicating a gene of interest? in vivo- transferring
the DNA to a host cell using a vector<div>in vitro- using PCR</div>
What are sticky ends, and why are they important? Exposed bases on either end
of both the gene of interest and the vector<div>These exposed bases can be used to
stick together the vector and gene of interest very easily, despite the DNA being
from two different organisms </div>
How do you make complementary sticky ends? Use the same restriction
endonucleases on both the vector and the gene of interest
How do you join the gene of interest and the vector together? Add DNA
ligases
How do you prepare the gene of interest for insertion into the vector? You add a
promoter at the start of the gene so that transcriptional factors can bind<div>You
also add a terminator so transcription stops at the appropriate point</div>
How does transformation work? You mix the vectors and bacteria cells in a medium
containing calcium ions to increase the permeability of the membrane <div>The
temperature is then rapidly increased and decreased to further increase the
permeability of the membrane, allowing the plasmid to enter</div>
What are the three outcomes of in vivo gene cloning? The plasmid does not take up
the gene of interest as the plasmid reseals itself<div>The plasmid takes up the
gene of interest but the bacterium cell does not take up the plasmid</div><div>The
plasmid takes up the gene of interest and the bacterium cell takes up the
plasmid <br></div>
What are gene markers used for? To identify bacteria cells that have
successfully taken up the plasmid containing the gene of interest
What are the three kinds of gene markers and how do they work? "Antibiotic
resistance- the vector plasmid contains two antibiotic resistant genes, and one is
cut out and replaced with the gene of interest. As such, the bacteria are first
placed on a plate with the antibiotic that the resistance gene is not cut out for,
thus eliminating bacteria that haven't taken up any plasmid. The two remaining
types of colonies are then pressed onto a plate with the second antibiotic, and the
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