Online Content Developed for Detecting Genetically Modified Organisms Biotechnology High School Kit

Online Supplement for GMOD Lab Kit

Online Content Developed for Detecting Genetically Modified Organisms
Biotechnology High School Kit

Neil E. Lamb, Ph.D. Director, Educational Outreach
HudsonAlpha Institute for Biotechnology
Huntsville, Alabama
nlamb@hudsonalpha.org

Project Summary/Description
The Hudson Alpha Institute for Biotechnology, with support from the U.S. Department of Labor
WIRED program, developed a series of biotechnology hands-on educational kits for use in high
school life science classes. Each kit was supported by an online supplement. This document
contains the online materials for the GMOD – Detection of Genetically Modified Organisms Kit
– the supplement can be found at http://www.hudsonalpha.org/education/kits/gmod

GMOD
Overview
This laboratory activity serves to explain what Genetically Modified Foods are and to provide an
exercise to detect the presence of genetically modified organisms (GMOs) in foods purchased
from the shelves of local grocery stores. It also allows students the chance to discuss ethical
issues associated with GMOs and their use in food.

In this experiment, students will test various foodstuffs purchased from a local store to determine
if they have been genetically modified. Students will first isolate DNA from samples of food and
will then use a scientific technique called polymerase chain reaction, or PCR, to amplify a
specific portion of the DNA; this will be followed by separating the DNA using a Flash Gel
system to determine whether their product does contain detectable GMOs.

What Are Genetically Modified Organisms?
As scientists discovered how genes function in the human and other various organisms, they have also
developed the technology of taking genetic material out of one organism and transferring it into another
organism in effort to express new traits that would not occur naturally.

One of the first examples of a genetically modified organism was in 1978 when the company Genetech
transferred the gene for human insulin into an E. coli strain. These E. coli cells were able to produce
insulin themselves which could be collected and used for diabetes treatments.

Since that time many other organisms have been genetically modified including other bacteria, mice, fish,
and plants.

The PBS website has an interactive dinner table activity:
http://www.pbs.org/wgbh/harvest/coming/coming.html.

Online Supplement for GMOD Lab Kit

  Click on the different foods on the table to learn about common foods that have been genetically
modified.

"Guess What's Coming to Dinner?" Nova Online. 2001. Public Broadcasting Service. 10 December 2008.
www.pbs.org/wgbh/harvest/coming/coming.html

Maize Chromosome Link
Below: An activity of a corn (maize genome). Clicking on regions of each chromosome will show you the
gene and physical trait located at that locus.

 “Putting DNA to Work - Maize Mutants.” Marian Koshland Science Museum of the National Academy of
Sciences. 2008. http://www.koshland-science-museum.org/exhibitdna/crops04activity.jsp

How Are GMOs Made?
To make a genetically modified organism, three main components are required: the gene you
want to transfer, the organism you want to put it into (target species), and a vector to carry the
gene into the target species cells.

The steps in making a GMO are relatively straightforward, but can be technically challenging.
The gene to be transfered (trans-gene) must be cut out and isolated from the original organism.
This is usually done by restriction enzymes, which are like molecular scissors, that recognize
specific sequences in the DNA and cut it at those places.
Restriction Enzymes

A restriction endonuclease is an enzyme that cuts strands of DNA at a specific point. It scans the
DNA for a specific target sequence, and when it finds that target sequence it cleaves the DNA.
Target sequences are relatively short. For instance, the common restriction enzyme EcoR1 only
has a 6 basepair target sequence. To date, thousands of restriction endonucleases (RE) have been
isolated, mostly from bacteria. Bacteria use these enzymes as a defense mechanism because the
can recognize and cleave foreign (virus) DNA.

Restriction endonucleases can cut double-stranded DNA in a few different ways. Sometimes it
cuts both strands at the same position, which causes blunt ends. Other times it cuts each strand at
a different point causing overhangs to occur. An overhang means that one strand is longer than

Online Supplement for GMOD Lab Kit

the other, and sometimes people refer to this as having sticky ends. See the diagrams below for
examples of blunt and sticky ends.

Restriction Enzyme Diagram

Below are two animations that illustrate how restriction endonucleases cleave DNA.
There is an online video of how restriction endonucleases cut DNA by McGraw-Hill Higher
Education: http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf

“Restriction Endonucleases.” Biology 7th Edition. 2005. McGraw-Hill Higher Education. 8 July
2009. http://highered.mcgraw-hill.com/olc/dl/120078/bio37.swf

The Dolan DNA Learning Center also has an online animation about restriction endonucleases
cut DNA: http://www.dnalc.org/ddnalc/resources/restriction.html.

Online Supplement for GMOD Lab Kit

“DNA Restriction.” DNA Learning Center. Cold Spring Harbor Laboratory. 12 December 2008.
http://www.dnalc.org/ddnalc/resources/restriction.html

The trans-gene is then inserted into a vector that is capable of getting inside cells of the target
species. To do this a scientist removes the portions of the virus’ genome that cause harm, but
leave the genes responsible for getting into the host cells. Then the target gene is inserted into the
host cells. Once in the host cell the genes will insert into the host’s genome. After this, every
time the genome is replicated and new cells are made the trans-gene will also be found the the
DNA of each new cell.
The PBS website has a good interactive activity about making a genetically modified crop:
http://www.pbs.org/wgbh/harvest/engineer/transgen.html.

“Engineer a Crop: Transgenic Manipulation.” Nova Online. 2001. 12 December 2008.
http://www.pbs.org/wgbh/harvest/engineer/transgen.html.

How to Test for a GMO
Most genetically modified crops have been "modified" using a common vector. In order to check
if a food has been genetically modified one can test for the presence of part of the vector's DNA
in the food of interest. The general procedure to do this is relatively simple using standard
molecular biology techniques.

Step 1:
   Extract the DNA from the food you want to test by mixing a small ground up piece of food
with an extraction buffer in a test tube. Grinding up the food breaks open the cells and the
extraction buffer separates the DNA from the rest of the cellular components.
Step 2:
   Amplification of the DNA - More DNA is required to run the GMO test than is naturally
found in a piece of food so the desired segment of DNA needs to be copied many times to
produce a larger volume of DNA for genetic testing. DNA amplification is the process in which
a sample of DNA is copied many times. This is done through a procedure known as the
polymerase chain reaction (PCR). In the polymerase chain reaction the DNA extracted from the
food is unwound and the two strands of the double helix are separated. The amplification
procedure selects just for the segment DNA we are testing for (the sequence of the most
commonly used GMO vector). In the reaction mixture there are many small fragments of DNA
that are complimentary to that target sequence. If the vector is present in the food's DNA, the
primers will bind and the vector DNA will be copied. This process continues many times
creating many thousands of copies of the specific segment of DNA.

  *In this experiment, if the food you chose was not genetically modified, its DNA would not
contain the vector genes in which the DNA primer would bind. Therefore the DNA would not be
copied. Go through the animation below to learn more about the PCR process.

Online Supplement for GMOD Lab Kit

There is a good interactive PCR activity on the Life Sciences Learning Center website of the
University of Rochester: http://lifesciences.envmed.rochester.edu/animation.html

“Polymerase Chain Reaction (PCR) - Virtual Lab.” Life Sciences Learning Center. University of
Rochester. 12 December 2008. http://lifesciences.envmed.rochester.edu/animation.html

Step 3:
  The last step is to visualize the DNA and see if the DNA has been amplified. This is usually
done by agarose gel electrophoresis.

   Gel electrophoresis separates segments of DNA based on charge and size. The DNA solution
is placed in a well of an agarose matrix. The matrix is then put into a box filled with a buffer
solution and hooked up to an electric current. This current causes one side of the gel to hold a
positive charge and the other side of the gel to have a negative charge.

   In the well, DNA is a negatively charged molecule due to the phosphate groups that constitute
the backbone of DNA. Therefore, by placing the DNA at the negative end of the gel matrix,
when the current is turned on the DNA will migrate down the gel towards the positive side
because the opposite charges attract.

   The agarose gel matrix is made up of a latticework of proteins, kind of like an obstacle course,
that the DNA must pass through. Because of this, the size of the DNA strand constitutes how
quickly it will move down the gel and how far it is able to go. Smaller segments will move more
quickly through the obstacle course and travel further down the gel.

  For this laboratory exercise the Flash Gel system will be used (pictured below). In this system
gel boxes will be supplied with wells already cut in them. A ladder solution will be pipetted in
one well and each DNA sample will be pipetted in subsequent wells. The gel boxes will then be
hooked up to a power supply to produce the electric current. This system is unique in that it is set
up so that you can see the DNA segments as they move down the gel. All the fragments of the
same size will move together and create a “band” appearance.

Online Supplement for GMOD Lab Kit

To determine the size of a DNA fragment a ladder should also be included in a separate well. A
ladder contains multiple DNA fragments of known sizes. Therefore, how far the sample
fragments traveled can be compared the ladder and the size of the sample DNA can be
determined. The ladder will produce many different “bands” or lines. Once the bands have begun
to approach the other side of the gel, the current is turned off. Now the bands in the sample wells
can be analyzed. If size is the unknown, the band can be compared to the bands in the ladder well
to determine approximately how many base pairs are in that DNA fragment.

   In this laboratory exercise different foodstuffs are being tested to see if they contain the vector
DNA segment. For each sample on the gel, one band will be present if the sample foodstuff is a
plant and another band will be present if the sample contains the GMO vector.

  Below is a sample gel result for this exercise. The different vertical columns are called “lanes”
with the well at the top where the DNA solutions are pipetted. Lane 1 is on the far left and
contains the DNA ladder. The lane on the far right contains the positive control sample
containing both a band signifying the DNA is from a plant and also a band signifying the DNA
has been genetically modified. The lanes between the ladder and the positive control contain
samples of DNA.

Online Supplement for GMOD Lab Kit

                                                                                                  l

In most laboratory-run gel electrophoresis experiments, another step must be taken in order to
visualize the bands on the gel. A stain, such as ethidium bromide, which inserts into the DNA
structure and fluoresces under UV light, is mixed with the DNA samples. Then, after the gel has
finished running, the gel must be placed under UV light, and the bands will appear glowing on
the darker gel. Below is the link to a good interactive animation about gel electrophoresis.

The Dolan DNA Learning Center has a good online interactive animation about gel
electrophoresis: http://www.dnalc.org/ddnalc/resources/electrophoresis.html.

“Gel Electrophoresis.” DNA Learning Center. Cold Spring Harbor Laboratory. 12 December
2008. http://www.dnalc.org/ddnalc/resources/electrophoresis.html.

What are the Ethical Issues?
Genetically modified organisms have been a hot topic of debate among environmentalists,
scientists, and policy makers. There are enormous benefits that can come from genetically
modified crops and animals, however there are also serious concerns about the consequences on
the environment and human health.

Online Supplement for GMOD Lab Kit

Some of the benefits of genetically modified crops are that genes can be inserted that cause the
plant to be resistant to many harmful things such as weeds, insects, and disease. This would
allow for less herbicides to be used on the crops and more crops to survive and produce food.
Crops can also be modified to have a longer shelf life in stores, grow in dryer/colder climates, be
resistant to pests, and even to have increased nutritional value.

The concerns regarding genetic modification deal with the possible unknown effects on human
health and the environment. Some new allergies have emerged due to the consumption of
genetically modified foods. A study in 1998 cited that a soybean allergy was developed in people
who ingested transgenic soybeans that were produced to be animal feed. Additionally, there are
other issues related to possible harmful side effects on other organisms that live with or depend
on the organism that is genetically modified. One example of this is Bt corn. While the
modification is helpful in making the corn toxic to caterpillars, the pollen from these modified
plants might also be fatal to the monarch butterflies. There are many issues to think about when
discussing the viability of genetically modified organisms. Look through the links below to read
more about the pro’s and con’s of genetic modification and decide for yourself.

Websites discussing the pro’s and con’s of genetically modified foods:
 http://www.pbs.org/wgbh/harvest/

  http://www.agriculture.purdue.edu/agbiotech/

Examples of GMOs
Bt corn
The European corn borer, Ostrinia nubilalis, has been known to cause major damage to corn
crops in the U.S. and Canada. The larval stage of this organism bores holes in the corn plants as
they feed. However, scientists discovered that there was a gene that they could insert into the
corn plant’s genome that would help them be resistant to these larva. The gene that is inserted
comes from the bacteria Bacillus thuringiensis and codes for an endotoxin protein that acts as an
insect stomach poison. The endotoxin proteins binds to cells in the intestinal lining of the larva
and the cells burst. This causes the larva to stop eating quickly and die within a few days
depending on how much toxin is ingested. While this protein is toxic to the European corn borer
larva, it is harmless and safe for most other organisms including humans. Genetically modified
corn with the Bacillus entotoxin, commonly called Bt corn, was introduced in 1996 and its use in
corn fields is expanding.

For more information about Bt corn, visit this website from the University of Minnesota:
http://www.extension.umn.edu/distribution/cropsystems/DC7055.html.

Golden Rice
Food scientists have also been applying genetic modification to crops with the goal of making
them more nutritious. This would be particularly beneficial in parts of the world where
nutritional deficiencies are common and a diet full of fruits and vegetables is not possible.
Vitamin A deficiency is thought to be responsible for at least 1 million deaths annually

Online Supplement for GMOD Lab Kit

worldwide. Golden rice has been developed in attempt to make rice more nutritious by inserting
genes that produce beta-carotene, a precursor to vitamin A. It is intended for this rice to be
grown and eaten by the poor and malnourished populations of the world and give them more of a
vitamin that is lacking in their diets. The beta-carotene in the rice causes it to have a
yellow/orange color instead of white, hence the name golden rice. The first prototype was
developed in 1999 and in 2004 the first golden rice was grown. Currently the genetically
modified rice is going through rigorous testing and assessment for safety and effectiveness
before it can gain approval for human consumption and widespread distribution.

To learn more about the golden rice effort, visit the Golden Rice Project website at:
http://www.goldenrice.org.

About HudsonAlpha
The Hudson Alpha Institute for Biotechnology is a not-for-profit research organization founded
in 2005. It is located in a spectacular 270,000 square foot building in Huntsville, Alabama. The
institute is a joint venture between private philanthropy and support from the state of Alabama.
Hudson Alpha aims to use biotechnology to improve human health, stimulate economic growth,
and inspire youth to seek careers in the field of science through educational outreach. The
institute is also committed to supporting teachers by providing professional development and
ongoing training based on new discoveries at the institute. For more information about
HudsonAlpha please see www.hudsonalpha.org.

NOTE: This program was funded by a grant awarded under the Workforce Innovation in Regional
Economic Development (WIRED) Initiative as implemented by the U.S. Department of Labor’s
Employment & Training Administration. The information contained in this product was created by a
grantee organization and does not necessarily reflect the official position of the U.S. Department of
Labor. All references to non-governmental companies or organizations, their services, products or
resources are offered for informational purposes and should not be construed as an endorsement by the
Department of Labor. This product is copyrighted by the institution that created it and is intended for
individual organizational, non-commercial use only.