Biotechnology:
“Or how I stopped worrying and
learned to love the sheep.”
Restriction Enzymes
• Restriction enzymes are compounds first
isolated in the 1970's
• They function by selectively cutting DNA at
specific sequences
Restriction Enzymes
• These cuts usually occur
in the following forms.
• The cut can be made
straight across a basepair sequence resulting in
a "Blunt End“
• The cut can be made in
an offset manner leaving
exposed nucleotide
sequences. These
exposed sequences are
called "Sticky Ends"
Blunt End
Sticky end
Gene Splicing
• The presence of
sticky ends allows
segments of DNA to
be joined together.
Since DNA strands
which have been cut
by the same
restriction enzyme
can easily bond
together according
to base pairing
rules.
Gene Splicing contd..
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This allows for genes to be "cut &
pasted" between organisms. This
can be seen with production of
human insulin.
The DNA sequence of insulin is
identified and cut out using a
restriction enzyme.
A plasmid from E. coli is removed
and cut open using the same
restriction enzyme
Since both fragments have
complimentary sticky ends the
bind and the gene for human
insulin is integrated into the
plasmid
The plasmid is then reinserted into
a bacterial cell. This cell will
produce insulin and is cultured.
Human insulin can now be
extracted and provided to
diabetics.
Gel Electrophoresis
• Gel electrophoresis
is a technique used
to separate
fragments of DNA.
• Separates fragments
as a function of size.
• Most types use
Agarose to separate
fragments.
• Agarose is a porous
gel. It can allow the
passage of
molecules through,
however, larger
molecules move
more slowly through
it since they cannot
squeeze through the
pores as easily as
smaller molecules.
Electrophoresis Apparatus
Electrophoresis Technique
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An agarose gel is casted with
several holes called wells at one
end.
The gel is placed in an
electrophoresis box which is filled
with an electrolyte buffer solution.
Samples of digested DNA are
placed in the wells
Electrical leads are attached to the
ends of the box creating an
electrical potential across the
apparatus.
Because DNA has a negative
electrical charge. It is "pulled"
towards the positive side of the
apparatus.
Also, since the smaller molecules
travel faster through the agarose.
Over time this separates the various
sized fragments of DNA.
The gel is then removed and
stained for DNA. This results in a
gel which shows several bands of
stained DNA.
Finished Gel
Gel Electrophoresis
DNA Fingerprinting
DNA is now a powerful tool in identification.
Based on the fact that the amount of "junk DNA" differs uniquely
between individuals.
Structural genes are often separated by large regions of repeating
basepairs.
The number of these repeats is unique to an individual.
Therefor when DNA from a person is cut with a restriction enzyme,
the length of the fragments will be unique to an individual.
DNA Fingerprinting Contd…
• This will therefor
produce a unique
banding pattern
following a gel
electrophoresis.
• This test is highly
accurate, and the
probability of another
individual possessing
an identical banding
pattern is estimated
as around
1:14,000,000,000.
DNA Fingerprinting
Cloning
Cloning: What it is
•
Cloning is the process of making a
genetically identical organism
through nonsexual means. It has
been used for many years to produce
plants (even growing a plant from a
cutting is a type of cloning). Animal
cloning has been the subject of
scientific experiments for years, but
garnered little attention until the birth
of the first cloned mammal in 1997, a
sheep named Dolly. Since Dolly,
several scientists have cloned other
animals, including cows and mice.
The recent success in cloning
animals has sparked fierce debates
among scientists, politicians and the
general public about the use and
morality of cloning plants, animals
and possibly humans
Dolly, the first mammal clone
Dolly: A Mammal Clone
• Dolly
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In 1997, cloning was revolutionized
when Ian Wilmut and his colleagues
at the Roslin Institute in Edinburgh,
Scotland, successfully cloned a sheep
named Dolly. Dolly was the first
cloned mammal.
Wilmut and his colleagues
transplanted a nucleus from a
mammary gland cell of a Finn Dorsett
sheep into the enucleated egg of a
Scottish blackface ewe. The nucleusegg combination was stimulated with
electricity to fuse the two and to
stimulate cell division. The new cell
divided and was placed in the uterus of
a blackface ewe to develop. Dolly was
born months later.