Bio Lab 12
What does this tell you about the GMO status of your food?
A band indicates that the food may be GMO-positive, the absence of a band indicates the food may be GMO-negative.
Explain why DNA fragments separate according to size in an electrophoresis gel.
DNA is negatively charged and is repelled by the negative electrode (cathode) and attracted by the positive electrode (anode) when an electric current is applied across the gel. It separates because different lengths of DNA move through the gel matrix at different rates. Longer fragments move more slowly than shorter fragments.
What molecules are present in the cell that might interfere with DNA extraction?
Enzymes, such as DNases, may degrade DNA. Metal ions act as cofactors and coenzymes for enzymes that degrade DNA. Cellulose plant cell walls may act as a barrier to DNA extraction.
How to performs this lab experiment?
First one must extract genomic DNA from food samples (a control non-GMO food and a grocery store food item) through several steps, but the screwcap tube must contain InstaGene matrix because it allows to extract DNA without degradation. Boiling the samples destroys these enzymes. After you centrifuge the samples to remove the InstaGene matrix and debris, the supernatant will contain intact extracted DNA. Then you will run PCR reactions to amplify GMO and natural plant sequences from the DNA. For this experiment, you will set up two PCR reactions for each DNA sample, which makes 6 PCR reactions in total. One PCR reaction, using the plant master mix (PMM), is a control to determine whether or not you have successfully extracted plant DNA from your test food. This is done by identifying a DNA sequence that is common to all plants by using primers (colored green in the kit) that specifically amplify a section of a chloroplast gene used in the light reaction (photosystem II). Why is this necessary? What if you do not amplify DNA using the GMO primers? Can you conclude that your test food is not GM or might it just be that your DNA extraction was unsuccessful? If you amplify DNA using the plant primers, you can conclude that you successfully amplified DNA, therefore whether or not you amplify DNA with your GMO primers, you will have more confidence in the validity of your result. The second PCR reaction you carry out will determine whether or not your DNA sample contains GM DNA sequences. This is done by identifying DNA sequences that are common to most (~85%) of all GM plants using primers specific to these sequences. These primers are colored red and are in the GMO master mix (GMM). Why do you have to set up a PCR reaction with DNA from certified non-GMO food? What if some GMO-positive DNA got into the InstaGene or master mix from a dirty pipet tip or a previous class? This DNA could be amplified in your test food PCR reaction and give you a false result. By having a known non-GMO control that you know should not amplify the GMO target sequences, you can tell if your PCR reactions have been contaminated by GMO-positive DNA. and in the third exercise you will electrophorese the amplified samples to visualize the DNA.
Why did you resolve your PCR products by electrophoresis?
Gel electrophoresis separates DNA molecules based on charge and size. After the bands are separated the gel is stained to visualize the band pattern. We can calculate the size of the DNA molecules, in base pairs, in each band.
Many foods containing GM crops are highly processed. Can you suggest how DNA from whole plants may differ from that extracted from processed foods, e.g., corn chips, cornmeal, etc.?
High temperatures or physical manipulation of the plant tissue during processing may destroy or fragment DNA.
What other information do you need to confirm the GMO status of your sample?
If there was a band in lane 4, we need to determine that there was not contamination of the samples to ensure the result is not a false positive. If there was no band in lane 4, we need to confirm that DNA was extracted from the sample and that the PCR reaction was functioning properly to ensure the result is not a false negative. Response below Not pertains to question The presence or absence of a 200 bp band in lane 5 indicates whether or not the test food contains GMOs. However, the validity of this result depends on the results from the other PCR reactions. The plant primers determine whether plant DNA was successfully extracted from the sample. The non-GMO food control is an indicator of false positive results, should they occur. If the non-GMO food control comes out as GMO-positive (showing a band in lane 2) it means that the PCR was contaminated at some point during processing. If your test food is also GMO-positive, you cannot trust this result. The GMO-positive template control is an indicator of false negatives. If the GMO-positive template control does not amplify, there is a problem with the PCR reaction and you cannot trust a GMO-negative result from your test food.
Why was the non-GMO food control prepared prior to your test food sample?
In the grinding process, airborne particles can travel through the air and contaminate samples of non-GMO foods. Also a mortar and pestle that is not properly washed can transfer minute sample. PCR only needs ONE molecule of DNA to make amplified product.
Why are you performing two PCR reactions on each DNA sample?
One reaction is a control to show we extracted plant DNA using primers to a universal plant DNA sequence. The second reaction is to identify the GMO target sequence.
In what organelles is plant DNA located?
Plant DNA is not only found in the nucleus, it is also found in the mitochondria and chloroplasts. Plants and other autotrophic organisms are the only organisms with chloroplasts. Plant DNA is more difficult to obtain intact because the cell wall must be destroyed.
What chemicals and molecules are needed for PCR, and what is the function of each component?
Taq DNA polymerase - a polymerase that is not sensitive to heat. It links the deoxynucleotide triphosphates to make a DNA strand that is complementary to the template • Deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP) - the basic units that are connected to make the complementary strand • Primers - short sequences of DNA that serve as beginnings of newly synthesized DNA. • Buffers and cofactors needed to make the reaction take place at an optimal rate.
What is the purpose of InstaGene matrix?
The InstaGene matrix is made of negatively charged microscopic beads that "chelate" or grab metal ions out of solution. It chelates metal ions such as Mg2+, which is required as a cofactor in enzymatic reactions. When DNA is released from your sample in the presence of the InstaGene matrix, the charged beads grab the Mg2+ and make it unavailable to the enzymes that would degrade the DNA you are trying to extract. This allows you to extract DNA without degradation. Boiling the samples destroys these enzymes.
What are the product band sizes?
The product band sizes in this lab are 455 bp for the plant primers and 200 bp for the GMO primers, but a 1% agarose gel is adequate to separate these bands despite their small size.
How can you test a food to find out if it contains material derived from a genetically modified organism (GMO)?
There are two methods to test for foods containing GMOs. The ELISA test is used to see if particular proteins are in a sample. PCR is used to amplify regions of GMO genomes.
Why do you also perform analysis on food that is known to be a non-GMO control?
To make sure samples have not been contaminated. It is also used as a comparison to show how a non-GMO banding pattern should look
Why do you need a molecular mass ruler alongside your samples?
We need a molecular mass ruler to calculate the size of each of our bands. We know exactly how many bands are in the ruler and the size of each of those bands. We can graph the size of the bands against the distance they moved in the gel to create a standard curve. We can then measure the distance our PCR product bands moved in the gel and use our standard curve to calculate the sizes of the product bands.
What is the purpose of the GMO positive control DNA?
We want to make sure our PCR reaction worked; if the positive control produces a positive result but I do not get a band in my test sample, the test is most likely non-GMO. If I do not get the 200 base pair band in the positive control, I can assume the PCR reaction did not work.
Did your test food generate a 200 bp band with GMO primer (lane 4)?
Yes or no
What methods can be utilized to detect GMO's and why they are used or not used?
it would be beneficial to be able to test foods found in the grocery store for the presence of GMO-derived products. This can be done in several ways. One would be to use an antibody-based test such as the enzyme- linked immunosorbent assay (ELISA), which can detect the proteins that are produced specifically by GM crops. However, the ELISA is not useful for testing foods that have been highly processed, because the proteins have most likely been destroyed and different GM foods produce different proteins. Another method is to use the polymerase chain reaction (PCR) to look for a DNA sequence common to GM foods. DNA is more resistant than proteins to processing and can be extracted from even highly processed foods. It is these GMO DNA sequences that we will be testing for in this laboratory.
