Methods and Concepts in the Life Sciences/Agarose Gel Electrophoresis
Agarose gel electrophoresis
Agarose gel electrophoresis is used to separate nucleic acids based on their length. By applying an electric field, the negatively charged nucleic acids move through an agarose matrix. The mobility of molecules is inversely proportional to the logarithm of their length, which means that small DNA or RNA fragments migrate faster than larger ones.
After the separation, the nucleic acids are stained and can then be viewed under UV light. By comparing the migration distance to a DNA size marker, it is possible to estimate the size of fragments. For use in further procedures, fragments can be extracted from the gel.
Agarose is a polysaccharide polymer material, generally extracted from seaweed. It is a linear polymer consisting of alternating D-galactose and 3,6-anhydro-L-galactopyranose linked by α-(1→3) and β-(1→4) glycosidic bonds.
Each agarose chain consists of approximately 800 monomers and the agarose polymer chains form helical fibres that aggregate into supercoiled structures. When solidified, the fibres form a three-dimensional mesh of channels of diameter ranging from 50 nm to >200 nm depending on the concentration of agarose used. The 3-D structure is held together with hydrogen bonds and can therefore be disrupted by heating back to a liquid state.
Agarose is commonly used in concentrations between 0.5 and 2 % (w/v). The concentration should be adjusted to the size of the expected fragments. For small fragments, the resolution is better at high agarose concentrations, whereas a lower concentration helps separate large molecules. Recommendations can be taken from the table below.
|Agarose concentration in % (w/v)||Efficient range of separation of linear DNA|
For small nucleic acids (10–1000 bp), sieving agarose with a concentration of 2–4 % can be used. Standard agarose has a gelling temperature of 35–42 °C and a melting temperature of 85–95 °C. Agarose may be modified by hydroxyethylation, and this substitution reduces the number of intrastrand hydrogen bonds, resulting in lower melting and setting temperature than standard agaroses. The exact temperature is determined by the degree of substitution, many available low-melting point (LMP) agarose types can remain as a fluid at 30–35 °C. This property allows enzymatic manipulations to be carried out by adding slices of melted gel with the DNA of interest directly to the reaction mixture.
The agarose polymer contains charged groups, in particular pyruvate and sulphate. These negatively-charged groups create a flow of water in the opposite direction to the movement of DNA in a process called electroendosmosis (EEO), and can therefore retard the movement of DNA and cause blurring of bands. Higher gel concentrations would cause higher electroosmotic flow. Low EEO agarose is therefore generally preferred for use in agarose gel electrophoresis of nucleic acids, but high EEO agarose may be used for other purposes.
The gel is prepared by dissolving the agarose powder in an appropriate buffer, such as TAE or TBE, to be used in electrophoresis. The agarose is dispersed in the buffer before heating it to near-boiling point. The melted agarose is allowed to cool sufficiently before pouring the solution into a cast as the cast may warp or crack if the agarose solution is too hot. A comb is placed in the cast to create wells for loading sample, and the gel should be completely set before use.
Once the gel has set, the comb is removed, leaving wells where DNA samples can be loaded. Prior to loading, the samples are mixed with a loading buffer, which contains a dense compound, such as glycerol or sucrose. This raises the density of the sample so that the DNA may sink to the bottom of the well. The loading buffer also includes colored dyes such as bromophenol blue and xylene cyanol, which are used to monitor the progress of the electrophoresis. The migration speed of bromphenol blue is similar to that of 200 bp fragments, whereas xylen cyanol runs at 4000 bp. Bromphenol blue is also a pH indicator: If it turns yellow, the buffer or the gel are not alkaline anymore and should be renewed.
During electrophoresis, the slab gel is completely submerged in buffer. This running buffer is the same as the buffer used for the gel. The most common types are Tris/Acetate/EDTA (TAE) and Tris/Borate/EDTA (TBE). TAE has a low buffering capacity, but it provides the best resolution for larger DNA and does not interfere with further processing of the DNA. Furthermore, it can be stored as a 50x stock solution, whereas TBE precipitates easily even at low concentrations. Borate is also problematic for further use of the DNA, however, TBE has a high buffering capacity.
EDTA is included in the buffers in order to inactivate nucleases which require divalent cations for their function.
DNA as well as RNA are normally visualized by staining with ethidium bromide, which intercalates between the bases of the DNA. When exposed to ultraviolet light, it will fluoresce with an orange color (605 nm). The fluorescence intensity increases drastically after binding to DNA, which is believed to be due to the hydrophobic environment between the base pairs, as water is a highly efficient fluorescent quencher. The ethidium bromide may be added to the agarose solution before it gels, or the DNA gel may be stained later after electrophoresis. Destaining of the gel is not necessary but may produce better images. The limit of detection for ethidium bromide is approximately 5 ng DNA per band.
Other methods of staining are available; examples are GelRed, SYBR Green, methylene blue, brilliant cresyl blue, Nile blue sulphate, and crystal violet. GelRed, SYBR Green, and other similar commercial products are sold as safer alternatives to ethidium bromide as it has been shown to be mutagenic in Ames test, although the carcinogenicity of ethidium bromide has not actually been established.
GelRed is structurally closely related to ethidium bromide: It consists of two ethidium subunits that are bridged by a linear spacer. The substance is marketed as more sensitive than ethidium bromide (0.1 ng DNA per band). SYBR Green requires the use of a blue-light transilluminator. DNA stained with crystal violet can be viewed under natural light without the use of a UV transilluminator which is an advantage, however it may not produce a strong band.
When stained with ethidium bromide, the gel is viewed with an ultraviolet (UV) transilluminator. Standard transilluminators use wavelengths of 302/312-nm (UV-B), however exposure of DNA to UV radiation for as little as 45 seconds can produce damage to DNA and affect subsequent procedures, for example reducing the efficiency of transformation, in vitro transcription, and PCR. Exposure of the DNA to UV radiation therefore should be limited. Using a higher wavelength of 365 nm (UV-A range) causes less damage to the DNA but also produces much weaker fluorescence with ethidium bromide. Where multiple wavelengths can be selected in the transillumintor, the shorter wavelength would be used to capture images, while the longer wavelength should be used when it is necessary to work on the gel for any extended period of time.
Following gel electrophoresis, DNA fragments are often isolated from the gel for use in further procedures. The first step is the identification of the desired band and its removal, for example using a razor blade. This should be carried out quickly to avoid damage to the DNA by the UV light. There are several methods for the isolation and purification of DNA from these bands, a common one is the use of kits, which are available from various manufacturers. Protocols included in these kits generally call for the dissolution of the gel-slice in 3 volumes of chaotropic agent at 50 °C, followed by application of the solution to a spin-column (the DNA remains in the column), a 70% ethanol wash (the DNA remains in the column, salt and impurities are washed out), and elution of the DNA in a small volume (30 µL) of water or buffer.