The study of Genetics is now becoming more and more important in everyday lives. Gene sequencing can help geneticists determine the probability of a patient carrying a gene-related disease, aid in the development of more drought-resistant crops, and many other things. However, gene sequencing is a very tedious job. Thankfully, there are modern techniques that help aid in gene sequencing, one of which is called gel electrophoresis.
DNA (short for deoxyribonucleic acid) is a polymer in the shape of a double-helix that is found in the nucleus of cells and is responsible for carrying genetic information. DNA is made up of nucleotides, which are nitrogen-containing purine pr pyrimidine bases linked to sugar (deoxyribose) and phosphate groups. The backbone of the DNA molecule is made of alternating sugar and phosphate groups. The bases are linked to the sugar parts of the DNA molecule. The purine bases are called guanine and adenine while the pyrimidines are called thymine and cytosine. Guanine always hydrogen bonds to cytosine and adenine always bonds with thymine. A single DNA molecule may contain millions of these hydrogen-bonded bases in specific pairs.
In electrophoresis, a scientist uses an electrical field to separate molecules based on their mobility. The rate at which the molecule moves is based on the size of the molecule as well as the charge of the molecule. Large molecules tend to move slower than small molecules. The medium in which the DNA molecules move through is a gel. Two different gels commonly used are polyacrylamide and agarose. Polyacrylamide is a polymer made of acrylamide and bisacrylamide and is synthetically made in a lab. Because acrylamide is a suspected carcinogen and a potent neurotoxin, agarose, which is a purified form of agar-agar that is extracted from seaweeds, is more commonly used. Polyacrylamide gels usually have a smaller pore size than agarose gels, which aids in better resolution of the DNA.
Agarose gels are often mixed at 0.5-2.0% concentration. Agarose at this concentration will separate a wide range of DNA fragment sizes, ranging from 200 base pairs to 50,000 base pairs long. Less agarose will separate large fragments of DNA better than this concentration, and more agarose will separate small fragments of DNA better, so the chemist needs to use his or her own judgment as to how much to use.
After measuring out the precise amount of agarose powder needed, a buffer solution is needed to dissolve the gel. As well as allowing the DNA molecules to run smoothly through the gel, the buffer solutions optimize the pH and ion concentration of the gel and bathe the gel while an electrical current, which moves the DNA molecules, is passed through it.
Two common buffer solutions used are TAE and TBE. The T stands for Tris (hydroxymethyl) aminomethane, which maintains the pH of the solution. The E stands for EDTA (ethylenediaminetetraacetic acid), which is another acronym for the solution that chelates divalent cations. This is important because some nuclei require divalent cations for activity, which may degrade the DNA sample while it is running trough the gel. The A stands for Acetic acid while the B stands for Boric acid. Both of these solutions provide the proper ion concentration for the buffer.
After mixing the agarose and the buffer together, the mixture needs to be boiled since agarose is not soluble at room temperature. The fastest way to do this is to put the mixture in the microwave for a few minutes. After the agarose powder is dissolved into the buffer solution, the mixture is poured into a gel casting apparatus, which consists of a tray, a support, and a comb. The tray is what molds the gel into its appropriate shape. The tray also has holes in the bottom that the support rests on top of. Because the gel is too flimsy, even after it has polymerized, it is pushed out of the tray by pushing the support through the hole. The comb’s job is to mold wells, in which the DNA is placed, while the electrical current is passing through it. The comb is put teeth-down into the gel before it has polymerized to mold wells into it.
Now that the gel has polymerized, it is put into a tank with a raised stage in the middle to put the gel. There are deeper reservoirs on both ends of the stage that holds the buffer solution. The same solution that was used to mix the agarose powder needs to be used. The gel is completely submerged while it is being run so the entire gel gets the same amount of electrical current. The electrical current is passed through the gel via two metal pins attached to the tank that are submerged into the buffer. One end is for the positive charge, called the anode, and the other is for the negative charge, called the cathode. Because DNA is negatively charged, the molecules will move toward the anode.
Now that the gel is prepared, there are certain steps that need to be followed to prepare the DNA. DNA from plant cells can be extracted by using various enzymatic procedures. The cell wall must be dissolved using a cell-wall digesting enzyme. After this enzyme is added, the cells are treated with a detergent and proteinase K to produce a high molecular weight of DNA molecules, which makes them suitable for electrophoresis analysis. Now that the DNA is released from the cells, the DNA molecules need to be cut in specific places using a restriction enzyme. Restriction enzymes are a defense mechanism against foreign invaders, such as viruses, for bacterial cells. Bacterial cells use these restriction enzymes to cut the invader’s DNA at specific nucleotide sequences to prevent its own DNA from being degraded. The chemist can choose one of the 200 known restriction enzymes to cleave the DNA sample in strategic places to make the molecules smaller and more able to be sequenced easily.
After letting the restriction enzyme digest the DNA for an hour at 37 degrees Celsius, the digestion needs to be stopped, or else the enzyme will continue to cut the DNA at random locations when it is brought to room temperature. This is done by adding EDTA to the DNA. EDTA stops this enzyme from working by denying it the divalent cations it needs to work, specifically the magnesium and calcium cations. Glycerol is then added to the sample to make the DNA sample more dense than the buffer that it is submerged in so the sample won’t float away. Now, the dyes (which are charged molecules) are added so we can monitor the DNA as it migrates across the gel. One dye is smaller than most the DNA fragments and will run as fast or faster than the smallest DNA fragments. The other dye is large and will travel along with the large DNA fragments. Assuming that our sample is somewhere between the dyes, the gel is stopped when the small dye gets near the end of the gel.
Now that the DNA samples are prepared, a micropipette is used to load the samples into the wells of the gel. A power supply is then attached to the metal pins on the tank, causing an electric current to run through the gel and moving the molecules across it.
When the DNA is finished running, the gel needs to be removed and exposed to ultraviolet light. We can make the DNA fluoresce under ultraviolet light by soaking the gel in an Ethidium Bromide solution. It is not the DNA itself that fluoresces, but the Ethidium Bromide. The Ethidium Bromide wedges itself into the spaces between the nucleotide bases of the DNA molecule and stays there. The more base pairs are in the sample, the more spaces are in it. Wherever her is DNA, a right band will be seen. When compared to a “ladder” (control), the amount of specific gene sequences can be determined.