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.
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, the question is whether a restriction enzyme has cut the sample DNA or not. The key to this answer is if the DNA has been cut there will be two or three bands produced by that DNA sample. This is because after the enzyme has cut the DNA there will be two different sized DNA fragments. If the patient has a mutation on both of his genes, two bands will result because all of the fragments will be cut. However, if the patient only has one copy of the mutation, that means that the other copy is normal and will not be cut. In this case the cut fragments will create two bands and the uncut fragments will create a third.
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. Lane 2 is normal control meaning that it contains DNA that is known to be normal and should not be cut if the procedure works correctly. Lane 3 contains DNA from a patient with mutations in both copies of their gene. Two bands are produced, both further down the gel than the band in lane 2. This makes sense because when the DNA is cut it produces two smaller segments. Lane 4 (right side) is a carrier who has one mutated copy and one normal copy. There are three bands, both of the bands from lane 3 due to the cut fragments as well as the same band as in lane 2 because of the uncut fragments.
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.