GMO Investigator Kit (BioRad)
Introduction
The purpose of this laboratory exercise is to test grocery store food products for the presence of
genetically modified organisms (GMO). The GMO investigator kit uses PCR to test for the presence of
two different GMO-associated sequences: the 35S promoter of the cauliflower mosaic virus (CaMV 35S)
and the terminator of the nopaline synthase (NOS) gene of Agrobacterium tumefaciens. One ot both of
these sequences are present in most of the genetically modified crops that are approved for distribution
in North America, Asia, and Europe
Procedure Background
Instagene Matrix
Instagene matrix consists of a suspension of negatively-charged Chelex® microscopic beads, which
bind divalent cations like magnesium. It is important to remove divalent cations from students genomic
DNA samples because cations assist enzymes that degrade the DNA template. The beads in the
Instagene Matrix quickly settle out of solution, so it extremely important that the suspension be
thoroughly mixed immediately prior to using. It is important that the beads be settled out prior to DNA
removal for PCR since the presence of them during PCR will inhibit the magnesium ions that are
required for Taq polymerase function.
Master mix
The master mix contains a mixture of dNTPs, buffer, magnesium ions, primers, and Taq polymerase.
For each sample, two separate PCR reactions will be set up. The first (green color coded) amplifies a
455bp region of the photosystem II chloroplast gene that is common in most plants. The second (red
color coded) uses “duplex” PCR – which menas two target sequences are simultaneously amplified. The
two pairs of primers amplify both the 203 bp CaMV 35S region, and the 225 bp NOS terminator
fragment. These primers have been included so that a greater range of GM food can be detected, since
some foods contain just the CaMV 35S promoter, some just the NOS terminator, and some both. By
using two sequences, about 85% of all GM foods currently available are detectable with this kit,
whereas CaMV 35S primers alone detect only ~70% of GM foods.
Mortars and Pestles
Please ensure that these are thoroughly washed to remove residual chemicals that may interfere with
PCR reactions. The student protocol calls for the preparation of a non-GMO food sample, and then wash
the mortar and pestle before preparing the unknown food sample. Since the non-GMO food is prepared
first, there should be no risk of contamination of the unknown. Postive-control DNA is provided to the
groups – DNA does not have to be extracted for this control.
Gel Electrophoresis
Orange G loading dye is added to each sample prior to gel loading. The loading dye contains glycerol,
which increases the density of the sample and ensures that it sinks into the well of the agarose gel. In
addition, the loading dye contains Orange G, that comigrates with DNA ~50bp in size on a 3% agarose
gel.
Due to the small size of expected bands, a 3% agarose gel is used.
Making DNA visible
Fast Blast stain contains positively-charged dye molecules that are attracted to and bind to the
negatively-charged phosphate groups of DNA molecules. A quick staining for 2 minutes is followed by
several de-staining rinses with warm water. Staining the DNA pinpoints its location on the gel.
Fast Blast stain will permanently stain your clothing – so dress accordingly!!!!!!!
Student Questions (typed answers are to be submitted as a GROUP. Include a copy of your
table)
Lane
1
Sample
Band appearance
(Yes,no)
Non-GMO food control with plant primers
Non-GMO food control with GMO primers
2
Test food #1 with plant primers
3
Test food #1 with GMO primers
4
Test food #2 with plant primers
5
6
7
Test food #2 with GMO primers
GMO positive control DNA with plant primers
GMO positive control DNA with GMO primers
8
1. Many foods containing GM crops are highly processed. Can you suggest how DNA from whole
plants may differ from that extracted from processed food, e.g., corn chips, cornmeal, etc.
2. What is the purpose of using InstaGene Matrix when extracting DNA?
3. Why was the non-GMO food control prepared prior to your test food samples?
4. Why are you performing two PCR reactions for each sample?
5. What is the purpose of the GMO-positive control DNA?
6. Explain why DNA fragments separated according to size in an electrophoresis gel.
7. Why do you need a molecular weight ruler alongside your samples?
8. What was your test food/s?
9. Did your test food/s generate a 200bp band with the GMO primer?
10. What does this tell you about the GMO status of your unknown?
11. How do the results of the other five PCR reactions help support or undermine your result for your
test food?