LABORATORY 6

Pre-lab Assignment for

PRACTICAL 6: Detecting genetically engineered corn

At the end of this pre-laboratory assignment you should be able to:

1. Define and explain the purpose of a genetically modified organism (GMO).
2. Detail the components and explain how the polymerase chain reaction (PCR) works.
3. Explain the principles of DNA electrophoresis.

Campbell Biology, Urry et al. (11th edition, Australian & New Zealand version)
Chapter 20 pp. 420-422, 438-439

Pre-laboratory assignment:
The pre-laboratory assignment is a MyLab and Mastering assignment. This assignment
should be completed before the start of the week in which Laboratory 6 is held (Week 11).
The assignment due date and time is Monday 11th October at 9 am in the morning. To
allow time to complete the assignment, please start it NO later than 8am on this day. As
part of good planning for learning, it is recommended you complete the assignment well
before Monday 11th October .

Preparation:
To prepare for the assignment, please read the information provided about the lab in the
following pages and the text book readings above. It is recommended you make notes on
the reverse side of this page about your reading.

To access the quiz:

Log in to CANVAS
> go to MyLab and Mastering
> go to assignments
> click on Genetics I ASSIGNMENT pre-lab 6 I Week 11
complete all questions in this assignment

If you have any questions about the questions inthis assignment, or if you encounter any
problems, please talk to the course coordinator, Mandy Harper in Room 101, Old Biology
building.

LABORATORY GUIDE BIOSCI 101

LABORATORY 6

BIOSCI 101 LABORATORY GUIDE

 LABORATORY 6

LABORATORY 6
Detecting genetically engineered corn

At the end of this practical you should be able to:

1. Describe the mechanism by which Bacillus thuringiensis (Bt) kills insect larvae.
2. Draw a diagram that shows how a DNA-based test can be used to identify a hybrid
between a genetically engineered (MON810) and a wild type corn variety.
3. Set up an electrophoresis chamber and pour an agarose gel.
4. Use a pipette to load a DNA ladder and PCR reactions onto an agarose gel.
5. Interpret the results of a DNA electrophoresis gel, and justify the need for positive
and negative controls in a PCR experiment.

Suggested reading:
Campbell Biology, Urry et al. (11th edition, Australian & New Zealand version) Chapter
20 pp. 420-422, 438-439

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LABORATORY 6

Genetic engineering

Genetic engineering is the in vitro alteration of DNA or RNA and the reintroduction of
the altered genetic material into a living organism. Genetic engineering has launched a
revolution in biotechnology and allowed the production of hormones, routine genetic
testing and the modification of microbes and plants.

Bacillus thuringiensis and its action

Bacillus thuringiensis (Bt) is a naturally occurring bacterium that has the ability to
produce a crystalline protein that is toxic to insect larvae. Upon ingestion by insect
larvae, Bt toxins cause gut cells to swell and burst, leading to larval death (Figure 1).
Commercial sprays making use of this bacterial property have been used for decades
by gardeners and organic farmers.

Different strains of Bt are selectively toxic to different insect orders. Different strains
target Lepidoptera (butterflies and moths); Diptera (flies and mosquitoes) and
Coleoptera (beetles). The Bt gene (Cry) has now been sequenced and genetically
engineered into crops including corn to provide insect resistance.

(Source: Vivian Ward, SBS)

Figure 1. The bacterium Bacillus thuringiensis (Bt), the Bt protein and its toxic effect
on insect larvae.

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 LABORATORY 6

Scenario

Recently, Sunshine Organic Foods (Ltd) became concerned that some plants on one of its
farms had been pollinated by the corn variety MON810. MON810 was being grown on
a neighbouring farm. MON810 is a genetically engineered variety of corn that contains
the Cry gene from the bacterium Bacillus thuringiensis. Concerned over public
perception, Sunshine Organic Foods employed GeneSleuth (Inc) – a company that
provides a commercial service for gene detection – to test their crops (Figure 2).

(Source: Vivian Ward, SBS)

Figure 2. Genetically engineered MON810 corn growing next to a Sunshine Organic

Foods (Ltd) crop.

GeneSleuth were given samples of 1000 plants from the Sunshine Organic Foods farm
plus control samples of MON810 and wild type corn. DNA was extracted from each
sample. The DNA was then amplified using the polymerase chain reaction.

One complication facing GeneSleuth was that Sunshine Organic Foods spray their corn
with Bacillus thuringiensis bacteria (which is organically accredited) and so the Cry gene
and the Cry protein could be present on all plants regardless of whether they were
genetically engineered or not.

To overcome the problem, GeneSleuth used a DNA-based test in which the two PCR
primers bound to the corn genome sequences flanking the engineered Cry gene. These
sequences are not present in the naturally occurring Cry gene. Corn is diploid and so
plants that have hybridised with the MON810 plants will have some kernels that have a
copy of the Cry gene and a copy of the wildtype sequence.

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LABORATORY 6

A PCR DNA-based test for the presence of the Cry gene

In diploid wild type corn, the GeneSleuth PCR primers amplify a 500 bp control
region of DNA on both chromosomes. When the amplified control region is run on
an electrophoresis gel the DNA migrates rapidly down the gel resulting in a single
band (Figure 3).

In hybrid Bt corn both a 500 bp control region and a 2500 bp region of DNA are
amplified. The 2500 bp region is a combination of the Bt gene and the control region.
When run on an electrophoresis gel the 2500 bp region migrates more slowly than
the 500 bp control region, resulting in two clearly defined bands (Figure 3).

If the PCR fails, for example if the DNA polymerase is inhibited, no bands will be
amplified. Therefore, a positive control is necessary in all PCR tests and contains a mix
of DNA from a MON810 and a wild type plant. A negative control is also necessary, in
order to detect DNA contamination. In this case the PCR reaction is run with water in
the place of sample DNA.

(Source: Vivian Ward, SBS)

Figure 3 The GeneSleuth DNA test for the Cry gene and the size of the resulting PCR
products.

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 LABORATORY 6

Electrophoresis of PCR reactions – Work in pairs

Setting-up the electrophoresis chamber & pouring an agarose gel

1. Put on gloves before starting.

2. Place the gel base in the chamber, then place the 'comb' in the gel base in the end
position. The gel base should be positioned with the seals (red ends) hard up against
the green sides of the gel chamber (Figure 4).

Figure 4. Electrophoresis gel chamber.

3. Get a Schott bottle of agarose from the water bath at the side of the laboratory
(careful it’s hot!) and gently swirl the contents before removing the lid. Pour all the
agarose into the base.
4. Allow 15 minutes for the agarose gel to set. Now realign the base containing the
gel with red ends facing the white ends of the gel chamber. The wells should be
positioned over the black stripe (Figure 5).
5. Once realigned, pour enough buffer to cover the gel (not beyond the max line)
into the electrophoresis chamber. Note that the gel should be covered by buffer.
6. Now remove the comb by gently pulling it directly upwards (Figure 5). The holes
('wells') that the comb leaves in the gel are where you will load your PCR reactions.
7. Before loading your PCR reactions wash wells using a disposable plastic pipette
provided, this helps your reactions load.

LABORATORY GUIDE BIOSCI 101

LABORATORY 6

Figure 5. Casting and pouring an agarose electrophoresis gel

Loading the PCR reactions onto your gel

1. Take a tube (each tube contains 10 μl of a DNA ladder or a PCR reaction).

2. Using the pipette, add 5 μl of the loading dye to the tube (15 μl total volume) and
flick the tube with your finger to mix the two solutions.
3. Tap the tube on the bench so the sample collects at the bottom.
3. Repeat for each of the 8 tubes (from now on use a new tip for loading each tube).
4. Using the pipette, take up all 15 μl of the first tube and insert tip through the buffer
and expel the contents into the first well in your agarose gel.
5. Start with tube 1 in the first well (DNA ladder), the positive control and negative
controls should be in well 7 and 8.
6. When all tubes are loaded, put the lid on the gel chamber. THEN ASK YOUR
DEMONSTRATOR TO CONNECT THE POWER SUPPLY
7. Set the timer for 30 minutes and press start.

Visualising the DNA & interpreting the results

1. Once your gel has run for 30 minutes, your demonstrator will turn off the power
supply and make sure that it is safe to remove your gel.
2. Take your gel in the tray provided and get the demonstrator to take a digital
picture of your gel. The demonstrator will print you a copy of your results which
you can now interpret.

BIOSCI 101 LABORATORY GUIDE

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Clean up instructions

1. Leave buffer in gel boxes. DO NOT EMPTY.

2. Place gel sample tubes, yellow tips and gels in the YELLOW BIOHAZARD BINS.

Hazardous substances

CHEMICAL HAZARD ADDITIONAL INFORMATION

Agarose Low risk

RedSafe DNA stain Non-carcinogenic but may cause
skin and eye irritations
Tris Harmful Harmful if swallowed or inhaled.
Causes irritation to skin, eyes and
respiratory tract

Sodium acetate Harmful May be harmful if swallowed/ inhaled.

Avoid contact with skin

EDTA Irritant Ethylenediaminetetraethanoic acid.

Skin, eye and respiratory irritant.

Glycerol Flammable Respiratory irritant in highly concen-

trated mist.

Orange G Irritant May be harmful if swallowed, inhaled

or absorbed through skin. Toxicology
not fully investigated.

Acetic acid Corrosive Avoid contact with eyes or skin

LABORATORY GUIDE BIOSCI 101

LABORATORY 6

BIOSCI 101 LABORATORY GUIDE