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Te c h n o l o g y
Genetic manipulation
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Understanding the ABC of DNA technology
How does gene technology really work? Over the next four issues Farming Ahead runs through the processes involved
in genetically modifying canola plants using a bacterial gene. From extracting deoxyribonucleic acid (DNA) from
bacteria, through to culturing the gene modified plants, these articles will go into the laboratory to show how it works.
by
Since bacteria are very simple cells, their
DNA is found in a jumble just inside the
cell membrane.
The bacteria used in this experiment are
natural soil-dwelling types.
This article shows the steps required to
extract DNA from a sample of bacteria.
Cristy Burne,
for CSIRO PLANT INDUSTRY
G
ene technology is continuing to provide
new options for agriculture and already
millions of farmers around the world are
growing some form of gene modified crop.
In this series of articles CSIRO opens the
doors of its laboratories to show the processes
involved in genetically modifying a canola
plant using a bacterial gene.
As gene technology continues to evolve and
more gene-tech crops become available,
decisions about which crops and products to
use will become more vital.
An understanding of the technologies
behind these new products can provide
farmers with the edge to take best advantage
of the options available.
Farmer workshops
CSIRO runs 1–2-day gene technology
workshops for farmers across Australia.
To find out more about gene technology contact
TJ Higgins on [email protected], or to access
more resources or information on the workshops,
contact CSIRO’s Ilaria Catizone on
[email protected] or phone
(02) 6246 5469.
Using a plastic pipette, fill a small centrifuge
tube with a sample of the bacterial suspension.
Place the lid on the tube.
Why extract DNA?
The tools of gene technology already
give breeders and biologists invaluable
information useful in tracking beneficial traits
and identifying disease-causing bacteria.
For example, these tools are in wide use
in plant and animal improvement in a
method called Marker Assisted Selection.
A newer use for gene technology is in
genetic manipulation, also known as GM
or transgenics. Here, a gene is added or
removed from an organism such as a plant
or animal.
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1a
3a
Photos: CSIRO
Extracting DNA
The first step in producing a gene modified
plant is to extract potentially useful DNA
from cells of interest, for example, cells taken
from another crop plant or soil-dwelling
bacteria. This means separating the tiny
molecules of an individual’s DNA from a
sample of their cells.
Every cell of an individual contains a
complete set of that individual’s DNA, so
almost any tissue can be used for extraction.
In the case of animals DNA is commonly
extracted from samples of body fluid, skin,
ear and even teeth.
In plants DNA can be extracted from
samples of leaf, root, flower, stem and fruit.
The aim of a DNA extraction is to separate
DNA from the rest of the sample.
In plant and animal cells the DNA is coiled
up inside the centre (nucleus) of every cell in
the sample and needs to be separated from
other parts of the cell such as cell walls, cell
plasma and tiny cell organelles.
2
1b
The bacteria first need to be cultured from a small
sample of a single pure culture. Simply add a
sample of the pure bacteria to a liquid growth
medium, and culture overnight at room temperature.
The bacteria use the nutrients in the growth medium
as food and will multiply rapidly. Different media
are available depending on the preferred diet of
the bacterial strain. Bacteria multiply by dividing:
each individual bacterium can produce two
more daughter bacteria. This leads to exponential
population growth, rapidly producing enough
bacteria for the rest of the DNA extraction. The liquid
culture now contains millions of bacterial cells
floating around in the growth medium. This bacterial
suspension is the source of the bacteria from which
DNA will be extracted.
3b
Place the tube in a centrifuge and spin for 30
seconds at high speed. As the tube is whirled
around, the bacterial cells separate from the
medium in the same way that tea leaves separate
from tea when whirling a billy. The cells are forced
to the bottom of the tube, forming a dense pellet of
bacterial cells.
FA R M I N G A H E A D
No. 169
February 2006
Genetic manipulation
Te c h n o l o g y
4
Remove the tube from the centrifuge and pour off
the liquid that remains at the top. This liquid is
called the supernatant, and consists of leftover
medium. It does not contain bacterial cells and
hence does not contain the target DNA. A pellet of
bacterial cells is now squashed at the bottom of
the tube.
9a
7
Mix the tube for five seconds by placing it on a
mechanical vortex. This quickly breaks the pellet
apart, helping each individual cell to contact the
lysozyme. Let the tube stand at room temperature
for five minutes to allow the lysozyme to act.
5a
5b
5c
5d
Add more bacterial suspension to the tube and
repeat steps 2 to 4. This creates a larger pellet at
the bottom of the tube, which in turn increases the
amount of DNA that can be extracted.
9b
8a
Centrifuge the tube for five minutes. Since the
partially pure DNA is lighter than the rest of the
tube’s contents, it will float to the top as a white
blob. Use a toothpick to remove the DNA blob and
transfer it to a clean tube.
6
So far the cells have simply been separated from
excess medium and have not been damaged.
To access the DNA inside, these cells need to be
broken open, in this case by using the chemical
scissors of an enzyme in buffer solution. The buffer
contains an agent to stabilise the pH, a detergent
to dissolve fats and to disrupt proteins in the cell.
Kick start the cell disruption by using a pipette to
add 0.25 millilitres of the buffer to the tube and
pump the pipette a few times to mix the buffer with
the pellet of cells. Add five microlitres of lysozyme,
an enzyme which works in the buffer to break down
the cell membranes to release their contents.
FA R M I N G A H E A D
No. 169
February 2006
8b
Place the tube in a boiling water bath for 40
seconds. The heat makes the proteins in the tube
unwind (denature), causing them to coagulate in
the same way that heating a raw egg causes the
runny egg ‘white’ to firm and change colour. As the
protein coagulates most of it is removed from the
DNA. After 40 seconds the white swirls of this
partially pure DNA mixture become visible.
10
Note that pure DNA is transparent. The white
colour of the blob indicates the DNA extracted is
still slightly contaminated with protein and pieces
of cellular debris. Additional purification is required
before using this DNA in further experiments.
This involves re-dissolving the DNA blob in water
and then adding alcohol slowly to cause the DNA to
separate out as a precipitate that can be lifted out
again on a toothpick and dissolved in water.
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