Download Genetic Engineering

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It is important to know the order of
nucleotides in a strand of DNA
The process of DNA sequencing (determining
the order of nucleotides) uses processes
found in vivo (inside the cell)
Recall that DNA polymerase adds
complementary nucleotides to a single strand
of DNA
The nucleotides are found in high
concentration in the cell
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Scientists attempt to mimic this process in
vitro (in a lab)
A length of DNA is first heated
◦ This separates the two strands
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Then, a heat-resistant DNA pol is added
Ordinary A, C, T and G nucleotides are added
in high concentration
However, a special type of each nucleotide is
also added in low concentration
Two features make them unique
1. They are fluorescently labeled
2. They lack two hydroxyl groups on carbon 2 and 3
(called dideoxy)
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The label allows them to be detected when
ran through a gel (more on this later)
The missing hydroxyls stops DNA pol from
adding any more nucleotides past that point
The addition of the modified nucleotides is
always random
Eventually, a set of labeled strands of all
lengths is generated
◦ Each ends with a dd nucleotide (one of four
possible colours)
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These are ran through a gel, arranging them
in order of size
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The gel is passed through a detector,
determining the order of the nucelotides
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Sometimes, scientists want to make copies of
a strand of DNA
◦ For example, a small amount of DNA left at a crime
scene
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This can be done in vitro through a
polymerase chain reaction
Involves a three step cycle
1. Heat to separate strands
2. Cooling to allow annealing of DNA primers
3. Addition of nucleotides via DNA pol
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http://www.youtube.com/watch?v=HMC7c2T
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http://www.youtube.com/watch?v=_YgXcJ4n
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Recall that restriction enzymes make “sticky
ends”
This works with any organism that has DNA
as its genetic material
As a result, sections of DNA from one
organism can be spliced into another
Bacteria is often used since it has plasmids
◦ These are small, circular strands of DNA
◦ Easy to remove
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The original version is called a cloning vector
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Plasmids are easily removed and reinserted
We have used these cloning vectors to
engineer bacteria to produce proteins such as
insulin and growth hormone
Recombinant
DNA
Gene for human
growth hormone
Gene for human
growth hormone
Human Cell
Bacterial
chromosome
Sticky
ends
DNA
recombination
Bacteria cell
Plasmid
Bacteria cell
containing gene
for human growth
hormone
DNA
insertion
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DNA that has been modified by adding
foreign segments is called recombinant
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It is also possible to transform multicellular
organisms such as plants and animals
To do so, microorganisms, such as bacteria
and viruses, must be used
For example, a bacteria that causes tumors in
plants can be altered so the tumor gene is
replaced by a useful gene
However, these methods can only work if the
microorganism can insert its DNA into a host
Inside plant cell,
Agrobacterium
Gene to be
transferred
Agrobacterium
tumefaciens
inserts part of
its DNA into
host cell
chromosome.
Cellular
DNA
Recombinant
plasmid
Plant cell
colonies
Transformed bacteria
introduce plasmids into
plant cells.
Complete plant
generated from
transformed cell.