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.
- 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.
- 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.
- 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.
|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
- 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.
- 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.