Download Molecular scissors slice DNA to isolate genes

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Te c h n o l o g y
Genetic manipulation
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Molecular scissors slice DNA to isolate genes
Gene technology allows scientists to work with molecules so tiny they cannot be seen and so complex they encode
important building blocks of life itself. This article opens laboratory doors to explain the methods and tools used to
produce genetically modified canola with beneficial traits. This is the second article in a four-part series.
Cristy Burne,
for CSIRO PLANT INDUSTRY
A
suite of genetically modified crops are
on the production line offering benefits
such as increased tolerance to pests, faster
growth or better grain properties.
The crops express traits unattainable
using conventional selective breeding
methods — they are produced by identifying
a beneficial trait in another organism,
isolating the gene (made of deoxyribonucleic
acid, DNA) controlling the trait and then
inserting the isolated gene into the plant
being modified.
The process requires scientists to sift
through thousands of genes in the search for
that short segment of DNA which might be
beneficial when inserted into a crop plant.
A typical plant contains about 30,000
different genes, each composed of a specific
sequence of nucleotide building blocks
organised in long DNA chains.
Bacteria (such as Agrobacterium, which is
the source of the Roundup Ready gene) have
about 5000 genes.
Having identified a gene of interest on a
particular DNA chain, the challenge then is to
cut the target section from the rest of the
DNA chain and paste it in to a useful spot in
the DNA of another organism. This is called
gene splicing.
Digesting DNA
The scissors used for this meticulous
task are natural enzymes, called restriction
enzymes, which slice through DNA molecules
in a process called ‘restriction digestion’.
The restriction enzymes are like DNA
scissors but can only cut in certain places.
Coding life
Everything living is created from
instructions encoded by DNA molecules.
Incredibly, these complex codes arise
from just four different molecules:
thymidine, cytosine, adenosine and
guanosine. Combinations of these four
chemicals encode all the proteins an
organism needs to survive.
Human DNA contains a sequence of
about three billion of these molecules
arranged in 46 chromosomes.
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The enzymes work by recognising very
short but specific DNA sequences within a
longer DNA sequence. They will cut through
DNA molecules wherever their trigger
‘recognition’ sequence occurs.
Each different restriction enzyme cuts in its
own distinctive place. Identical strands of
DNA will be cut in identical places, thus
forming matching sets of DNA fragments.
Plasmid DNA
Plasmids — small loops of DNA usually
found in bacteria — replicate by inserting
themselves into bacteria where they multiply
(to 100 copies or more per cell), so when
the bacteria replicate, the plasmid DNA is
replicated at the same time. This trait
makes plasmids very handy when creating
multiple copies of a useful DNA segment or
gene (gene cloning).
If a useful gene can be cut from its
original DNA strand and inserted into a
plasmid DNA, that gene then can be
replicated automatically.
1a
Cut and paste
Restriction enzymes make pasting a useful
gene into a plasmid DNA relatively simple.
Most restriction enzymes break DNA using
jagged cuts, so they create DNA segments
with serrated ‘sticky’ ends.
If the same restriction enzyme is used
to digest both the DNA containing the
useful gene and the plasmid DNA, the
segments produced will have matching
sticky ends, allowing the different DNAs
to mesh and form a single DNA. This is
called ‘ligation’.
Ligation sticks two DNA molecules
together. The binding process is carried out
by DNA ligase, an enzyme which chemically
sews DNA segments together. Cells use DNA
ligase naturally when repairing breaks in
their own DNA.
Put it to the test
The following laboratory protocol shows
how to isolate a useful gene from a bacterium
and then insert this gene into a plasmid DNA.
The process is achieved by first digesting the
bacterial DNA using a restriction enzyme;
then separating the DNA fragments to select
the gene of interest; and finally adding this
gene to a plasmid DNA digested using the
same restriction enzyme.
The experiment requires the
bacterial DNA prepared using the
method explained in Farming Ahead
No. 169, page 28.
1b
Photos: Cristy Burne
by
1c
Use a micropipette to add two microlitres (µL) of the
buffer into two tubes, one containing the bacterial
DNA and the other containing plasmid DNA.
The buffer contains an agent to stabilise the pH and
ensure a stable working environment for the
restriction enzyme. Restriction enzymes, like all
enzymes, are tightly folded proteins that will only work
in certain environments. If conditions such as pH or
temperature become too extreme, the protein will
start to unwind (denature) and can no longer function.
FA R M I N G A H E A D
No. 170
March 2006
Genetic manipulation
Te c h n o l o g y
6a
4a
2
Add 2µL of restriction enzyme to the bacterial DNA
tube. The restriction enzyme in this case is EcoRI,
which recognises the DNA sequence ‘GAATTC’ and
makes a jagged cut through the DNA chain wherever
this sequence occurs. The letters of the sequence
code for the four nucleotides from which all DNA is
composed: adenosine, guanosine, thymidine and
cytosine (see box on page 20 on coding life).
6b
4b
Incubate the tube at 37 degrees Celsius for one
hour to allow the enzyme to react with the DNA.
The enzyme will cut through the DNA wherever it
can identify its trigger recognition sequence.
Incubation at this temperature optimises the
reaction: at a cooler temperature the enzyme will
not cut as well; at a warmer temperature it will start
to unwind and lose activity.
3a
5
Load about 18µL of the digested DNA into one end
of a gel. Gels form the basis of a separation
method called ‘electrophoresis’, which separates
individual DNA fragments from a jumbled sample
by using the properties of a jelly-like gel.
Electrophoresis is like a running race for DNA: it
works by passing a current through a gel loaded
with DNA fragments at one end. Since DNA has a
negative charge, the current causes the DNA
segments to move through the gel toward the
positively charged opposite end. Different DNA
fragments move at different rates, depending on
their size, charge and gel type. This means some
fragments race ahead and others fall behind. By
the time the first fragment has reached the end of
the gel, the field of fragments will have spread out
along the length of the gel, allowing individual
fragments to be identified. When the target gene
has been identified in this way, it simply can be cut
out of the gel.
7a
In the next step the digested DNA fragments will
be separated to identify the target fragment
containing the useful gene and that the plasmid
DNA is digested. Add 5µL of DNA dye to the tube
and mix. The dye will move with the DNA fragments,
making them visible.
3b
Farmer workshops
3c
Give the tube a flick to mix the contents. Place it
in a microfuge. The microfuge spins the tube to
ensure a uniform mix.
FA R M I N G A H E A D
No. 170
March 2006
CSIRO runs 1–2-day gene technology
workshops for farmers across Australia.
For more information about gene technology
contact TJ Higgins on [email protected] or
for information on workshops contact Ilaria
Catizone on [email protected] or phone
(02) 6246 5469.
7b
The digested plasmid DNA is ligated to bacterial
DNA using DNA ligase to reform a new, larger
plasmid loop, which now contains extra, introduced
DNA including the target gene.
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