PCR to Detect
Genetically Modified Organisms (GMOs) Field Trip
Background Information
A
genetically modified organism (GMO) is when an organism’s genetic
make-up has been changed using recombinant DNA technology, to
produce something non-native to the species. There are many
examples of GMOs including transgenic animals, fish, plants, and
microorganisms. A specific example of genetic modification would be
the introduction of the human insulin gene into E.coli to
produce human insulin. Today GMOs are being developed and used
world-wide; in the United States most of our corn and soybeans have
been genetically modified.
Farmers have
been using genetic modification in crops for thousands of years. In
early farming, seeds from plants that produced good crops were saved
and used the following season. Also, cross-bred plants or “hybrids”
have been developed for many types of crops. These plants are not
considered GMOs, because they are naturally manipulated, the
introduction of the new gene or trait does not require recombinant
DNA techniques. To create a GMO there are many methods of
introducing a new gene into a plant. The most common recombinant
DNA technique utilizes Agrobacterium, a soil bacteria, and a
genetic promoter from the cauliflower mosaic virus (CMV). We will
use the Polymerase Chain Reaction in this lab to detect whether the
food samples (corn chips and corn meal) have been genetically
modified by amplifying the 35S promoter region of the CMV.
In 1983 Kary
Mullis, a scientist at the Cetus Corporation in California, imagined
a way to replicate (copy) DNA in the lab. He worked on the idea for
two years, and in 1985 published and filed a patent for his idea. In
1993 he won the Nobel Prize in Chemistry for his work developing the
Polymerase Chain Reaction. The Polymerase Chain Reaction (PCR) is
now a very widely used technique for copying DNA. Starting with only
a small sample of DNA, PCR can generate many copies of a specific
DNA segment to be used for further analysis. This process is also
called DNA amplification. PCR has revolutionized molecular biology,
and is now routinely used in biological research, forensics
(criminal investigations), medical testing, and anthropology.
During the
PCR-GMO Field Trip, students will isolate DNA from food, set-up and
perform a PCR reaction, learn the underlying principles of PCR, and
analyze their results on an agarose gel.
PCR
utilizes:
·
template DNA -
the starting DNA of interest.
·
two primers (oligonucleotides)
- short, single-stranded, synthesized pieces of DNA that complement
sequences on each side of the region of the template DNA that is
being amplified.
·
thermostable
DNA polymerase - typically Taq (Thermus aquaticus), a
heat stable enzyme capable of adding nucleotides to a growing DNA
strand.
·
dNTPs - a
supply of the 4 nucleotides needed to make the new DNA strands.
·
magnesium - a
cofactor for the polymerase.
·
a buffer
solution - to maintain the pH and salt concentrations appropriate
for the polymerase
Once these
components are combined they go through a series of temperature
changes (cycles), repeatedly, in a machine called a thermal cycler.
This process will generate exponential copies of the DNA segment of
interest. In other words, if you start with 2 copies of the DNA
segment of interest, after 20 cycles you will theoretically have
2(nth) = 2(20th) = 1,000,000 copies of that segment.
Each cycle
consists of three parts: denaturation (D), annealing (A), and
elongation or extension (E). Denaturation separates the
double-strands of the DNA molecule at a relatively high temperature
of 90-96°C,
annealing allows the primer sequences to match and bind to the
flanking regions of the target area at a moderate temperature
between 40-70°C,
and elongation or extension occurs as the polymerase adds
nucleotides to the growing strand at 68-72°C.
The initial denaturation may last from 2-5 minutes, then is
typically 30 seconds during subsequent cycles. Annealing and
elongation steps are typically 1 minute each, with a final
elongation that may last up to 10 minutes. After the cycling is
complete the PCR product is held at 4°C.
The number of cycles, temperatures and time lengths are programmed
into the thermal cycler, which is somewhat like a computerized heat
block.
Since the
anticipated product length is known, PCR products can be evaluated
using an agarose gel when run alongside a DNA size standard, or
marker, with DNA bands of known sizes. Template DNA will be longer
in length then the desired PCR product, run more slowly through the
gel, and appear as a band closer to the top of the gel. Unbound
primers or “primer dimers” will be shorter, run faster, and create
smears of DNA closer to the bottom of the gel. The amplified DNA in
the PCR field trip ranges in size from 180 to 1500 base pairs in
length, the primers are 22 and 24 base pairs in length.
PCR has
become part of the popular culture. “Jurassic Park” and “CSI” are
just two examples where PCR is crucial to the plot. Some fun PCR
facts to share with your students*: …PCR has been used to amplify
DNA from…
·
poached moose
meat in hamburger
·
a preserved
quagga (an zebra relative that became extinct 100 years ago)
·
crime scenes
·
eight-celled
human preembryos, to detect cystic fibrosis
·
the brain of a
7000 year old American mummy
·
patients for
disease diagnosis
*Life Third
Edition, Ricki Lewis, WCB McGraw-Hill, 1998.
You can also
find some useful information on the web about PCR, including
animated computer tutorials. One tutorial sponsored by Cold Spring
Laboratory can be found at:
www.dnalc.org/shockwave/pcranwhole.html.
Please note that this requires a Shockwave Plug-in to view, you will
be prompted on how to download this plug-in when you visit the site.
For more
information on GMOs check out the following internet links
http://en.wikipedia.org/wiki/Genetically_modified_organism
http://www.newscientist.com/channel/life/gm-food
http://www.organicfooddirectory.com.au/gmo.php
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