In the olden days, DNA fragments were laboriously separated by using gravity. In the 1970s, the powerful tool called DNA gel electrophoresis was developed, in which electricity was used to separate DNA fragments by size as they migrate through a porous gel matrix.
Gel electrophoresis is now used to sort out strands of DNA, RNA or protein molecules by size, by using agarose gel and electrical current. This is achieved by moving negatively charged DNA molecules through an agarose matrix to which an electric field is applied (electrophoresis). Shorter molecules move faster and migrate farther than the longer ones, thus separating them by size. DNA Gel electrophoresis is generally used after amplification of DNA using PCR technique. It is done in the following steps:
- To make agarose gel, put a little agarose into a flask and add buffer solution to it. The buffer aids the electric current to travel through the gel. Heat the solution until agarose melts in the buffer and then pour the material into the gel mould. Place the comb in mould to make wells in the gel in which DNA can later be placed. When the gel solidifies, carefully remove the comb.
- Pour buffer into the electrophoresis box. The buffer conducts the electric current and keeps the gel from drying out.
- Place the gel mould inside the electrophoresis box, barely submerged in the buffer.
- Add loading buffer that contains dye to the DNA sample.
- Use micropipette to load DNA sample mixed with the loading buffer into the first well of the agarose gel.
- Use micropipette to load DNA size standard into the next well of the agarose gel. The DNA size standard contains strands of known length.
- Connect the electrophoresis box to the electric current. The negative (black) end of the current must be placed on the end closest to the wells.
- Turn on the electric current. The DNA strands move away from the negative current. The short strands move through the gel more quickly than the long strands. This can be observed due to the blue dye of the loading buffer.
- Turn off current and remove the gel mould from the electrophoresis box.
- Place gel in DNA staining solution, ethidium bromide. This binds to DNA and can be viewed under fluorescent light.
- Remove gel from ethidium bromide after about 30 minutes and place it on UV light box. The DNA bands will be visible for both the DNA sample and DNA size standard.
Some details related with gel electrophoresis are mentioned below.
An electrophoresis chamber or box in which gel and buffer can be placed and electric current can be passed so that DNA strands begin moving through the gel.
Gel casting trays are available in a variety of sizes and are made of UV-transparent plastic.
Sample combs around which molten agarose is poured to form sample wells in the gel.
Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to “fall” into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.
Ethidium bromide is a fluorescent dye used for staining nucleic acids so that they become visible.
Transilluminator is an ultraviolet light box, which is used to visualize ethidium bromide-stained DNA in the gel.
To prepare gel, agarose powder is mixed with electrophoresis buffer to the desired concentration and then heated in a microwave oven until completely melted. Most commonly, ethidium bromide is added to the gel (0.5 ug/ml) to facilitate visualization of DNA after electrophoresis. After cooling the solution to about 60oC, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature.
After the gel has solidified, the comb is removed. The gel, still in its plastic tray, is inserted horizontally into the electrophoresis chamber and just covered with buffer.
Samples containing DNA, mixed with loading buffer, are then pipetted into the sample wells, the lid and power leads are placed on the apparatus and a current is applied. You can confirm that the current is flowing by observing bubbles coming off the electrodes. DNA will migrate towards the positive electrode, which is usually coloured red.
The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes. Bromophenol blue and xylene cyanol dyes migrate through agarose gels at roughly the same rate as double-stranded DNA fragments of 300 and 4000 bp, respectively.
When adequate migration has occurred, DNA fragments are visualized by staining them with ethidium bromide, which is often incorporated into the gel so that staining occurs during electrophoresis, but the gel can also be stained after electrophoresis by soaking in a dilute solution of ethidium bromide.
To visualize DNA or RNA, the gel is placed on an ultraviolet transilluminator.
Migration of DNA Fragments in Agarose
Fragments of linear DNA migrate through the agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. In other words, if you plot the distance from the well that DNA fragments have migrated against the log10 of either their molecular weights or number of base pairs, a roughly straight line will appear.
Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass. Typically, uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two topologically different forms of DNA, corresponding to super coiled forms and nicked circles.
Agarose Concentration
By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilitate separation of small DNA fragments, while low agarose concentrations allow resolution of larger DNA fragments.
Voltage
As the voltage applied to a gel is increased, larger fragments migrate proportionally faster than smaller fragments. For that reason, the best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel (the cm value is the distance between the two electrodes, not the length of the gel).
Electrophoresis Buffer
Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel. Conversely, if you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it.
Effects of Ethidium Bromide
Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels. It can be incorporated into agarose gels or added to samples of DNA before loading to enable visualization of the fragments within the gel. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility.