Testing for Genetically Modified Organisms Through the Use of PCR

   Background

What are GMOs?

GMOs, or genetically modified organisms, are animals or plants that have had traits added or amplified for human benefit. Genetic modification can be as simple as selective breeding of a species in order to bring out a quality. However, in recent years it has become possible to insert a gene from one species directly into another species. The most common uses for GMOs are in the agriculture business. Crops are often modified to improve features such as frost, pest, or drought resistance, to give plants longer shelf life, or to improve appearance and taste.

How are They Made?

The GOI (gene of interest) is identified in one species and isolated. This gene is placed in a plasmid, which is inserted into an agrobacteria. The agrobacteria is then inserted into a plant cell. The cell sees the inserted gene as one of its own, and the plant will develop with the specified trait.

Identification

There are two methods used to identify GMOs: ELISA and PCR. ELISA (enzyme-linked immunosorbent assay) identifies genetically modified organisms by identifying specific proteins. The disadvantages of this method are that the produce being tested must be fresh, and each test must be specific to a certain crop. However, ELISA is inexpensive and relatively simple. PCR (polymerase chain reaction), on the other hand, identifies all sequences of DNA foreign to the plant. Because a relatively small number of DNA sequences are inserted into crops, it is easy to test for many at once (a PCR test can identify up to 85% of all GM crops).

Controversy

GMOs have both advantages and disadvantages. Positive effects include the reduction of pesticides and herbicides in agriculture, and the possibility of nutritional value and reduced stress on farmland. However, opponents of GMOs warn that "superweeds" and "superpests", no longer affected by resistant crops, could develop. Other fears include reduction of plant diversity and potential allergic reactions resulting from crossing species. Although there are requirements in Europe to label genetically modified foods, there are no such laws in the United States due to the influence of agribusiness lobbies.

Procedure
1. Put 1 gram of cornflour into a mortar. Add 10 ml of distilled water, and grind into a pipetable mixture. Repeat with the broccoli. This is done to break down the cell wall.

The cornflower supplied in this lab is a non-GMO control. It verifies that the test will not give results for non-GMO foods with GMO primers.

2. Pipet 50 μL of the cornflour mixture and the broccoli into their respective tubes (labeled "non-GMO" and "test"). Add 50 μL of InstaGene to each tube.
3. Place the tubes in a 95°C water bath for five minutes. This breaks the cell and nuclear membranes. Afterwards, centrifuge the tubes.

The InstaGene added in step two prevents DNAse in the cytoplasm from destroying cell DNA that is released once the nuclear membrane is broken.

4. Prepare six test tubes. The contents should be as follows:

1. 20 μL non-GMO control (cornflour), 20 μL plant master mix
2. 20 μL non-GMO control, 20 μL GMO master mix
3. 20 μL test food (broccoli), 20 μL plant MM
4. 20 μL test food, 20 μL GMO MM
5. 20 μL GMO positive control, 20 μL plant MM
6. 20 μL GMO positive control, 20 μL GMO MM

The master mixes are primers which will search for a specific sequence of DNA. Because tubes one, three, and five contain plant primers, they should not show up as bands after the PCR testing. Tube two contains GMO primers, but the food is guaranteed to be non-GMO. If a band shows up from the test, this indicates contamination or some other failure in the experiment. On the other hand, tube six must produce a band, because it contains the GMO-positive control with GMO primers. A lack of a band would also mean a failure in the experiment. Tube four is the only one with completely unpredictable results.

5. Place tubes in the PCR machine (thermal cycler). The PCR machine uses fluctuating temperatures to replicate DNA.

6. Load 20 μL of each sample into the gel electrophoresis apparatus and run the gel for 3 minutes at 200 V.

Results

Bands appeared in lanes 2, 4, and 6. This means that the test food, broccoli, is genetically modified. There is an extra band in lane 2, the cornflour, which is likely due to contamination during the procedure.