Agarose gel electrophoresis-Electrophoretic Materials

    

Agar is a natural polysaccharide mainly composed of agarose (about 80%) and agarose gel. Agarose is a neutral substance composed of galactose and its derivatives, without charge, while agarose gel is a strong acidic polysaccharide containing sulfate and carboxyl groups. Due to the charge of these groups, it can produce strong electroosmotic phenomena under the action of an electric field. In addition, sulfate groups can interact with certain proteins, affecting the electrophoresis speed and separation efficiency. Therefore, currently agarose is commonly used as the electrophoresis support for plate electrophoresis, with the following advantages:

1) Agarose gel electrophoresis is simple in operation and fast in speed. Samples can be electrophoresis without prior treatment. 

2) Agarose gel has uniform structure, high water content (about 98%~99%), approximate free electrophoresis, and very little adsorption on samples, so the electrophoretic pattern is clear, with high resolution and good repeatability.

3) Agarose is transparent without UV absorption, and the electrophoresis process and results can be directly detected and quantified using UV lamps.

4) After electrophoresis, the zone is easy to stain, the sample is easily eluted, and it is convenient for quantitative determination. It can be made into a dry film for long-term storage.

Currently, agarose is commonly used as an electrophoresis support to separate proteins and isoenzymes. The combination of agarose electrophoresis and immunohistochemistry has developed into immunoelectrophoresis technology, which can identify complex systems that cannot be identified by other methods. Due to the establishment of ultra trace technology, 0.1ug of protein can be detected.

    

Agarose gel electrophoresis is also commonly used to separate and identify nucleic acids, such as DNA identification, DNA restriction endonuclease mapping, etc. Due to its convenient operation, simple equipment, low sample size, and high resolution, this method has become one of the commonly used experimental methods in genetic engineering research.

The separation of nucleic acids by agarose gel electrophoresis is mainly based on their relative molecular weight and molecular configuration, and is also closely related to the concentration of gel.

1) The relationship between nucleic acid molecule size and agarose concentration

A. The size of DNA molecules In gel, the migration distance (mobility) of DNA fragments is inversely proportional to the logarithm of base pairs. Therefore, the size of unknown fragments can be measured by comparing the migration distance of standards with known size with that of unknown fragments. But when the DNA molecular size exceeds 20kb, ordinary agarose gel is difficult to separate them. At this time, the mobility of electrophoresis is no longer dependent on the molecular size. Therefore, when using agarose gel electrophoresis to separate DNA, the molecular size should not exceed this value.

B. The concentration of agarose is shown in the following table. DNA of different sizes needs to be separated by electrophoresis with agarose gel of different concentrations.

The order of movement speed of DNA with different configurations is: covalently closed circular DNA (cccDNA)>linear DNA>open double stranded circular DNA. When the concentration of agarose is too high, circular DNA (generally spherical) cannot enter the gel, and the relative mobility is 0 (Rm=0), while linear double stranded DNA (rigid rod) of the same size can move forward in the long axis direction (Rm>0). It can be seen that the relative mobility of these three configurations mainly depends on the concentration of gel, but at the same time, it is also affected by the current strength, buffer ion strength, etc.

Agarose gel electrophoresis for separating nucleic acids can be divided into vertical type and horizontal type (plate type). In horizontal electrophoresis, the gel plate is completely immersed in the electrode buffer solution for 1-2 mm, so it is also called submersible type. At present, the latter is more used because it is convenient for gel preparation and sample addition, simple electrophoresis tank, easy to make, and can prepare gel plates of different specifications according to needs to save gel, so it is more popular.

When lacking ions, the current is too small and DNA migration is slow; On the contrary, buffer solutions with high ionic strength can generate a large amount of heat due to excessive current, and in severe cases, can cause gel melting and DNA denaturation.

Common electrophoresis buffers include EDTA (pH 8.0) and Tris acetic acid (TEA), Tris boric acid (TBE), or Tris phosphate (TPE), with a concentration of approximately 50mmol/L (pH 7.5-7.8). Electrophoretic buffer is usually prepared as a concentrated stock solution and diluted to the desired multiple before use. TAE has lower buffering capacity, while the latter two have sufficiently high buffering capacity and are therefore more commonly used. Long term storage of TBE concentrated solution may result in precipitation. To avoid this drawback, store 5 times the solution at room temperature and dilute it 10 times with 0.5 times the working solution to provide sufficient buffering capacity.

C. The preparation of gel takes diluted electrode buffer solution as solvent, uses boiling water bath or microwave oven to prepare a certain concentration of sol, fills it with horizontal rubber frame or vertical rubber film, inserts comb, and naturally cools it.

Dissolve the DNA sample in an appropriate amount of Tris EDTA buffer, which contains 0.25% bromophenol blue or other indicator dyes, and 10% -15% sucrose or 5% -10% glycerol to increase its specific gravity and concentrate the sample. To avoid the possibility of U-shaped bands in electrophoresis results caused by sucrose or glycerol, 2.5% Ficoll (poly sucrose) can be used instead of sucrose or Glycerol.

The experimental conditions of separating macromolecular DNA by agarose gel showed that the separation effect was better at low concentration and low voltage. Under low voltage conditions, the electrophoretic mobility of linear DNA molecules is proportional to the voltage used. However, as the electric field strength increases, the increase in migration rate of larger DNA fragments is relatively small. Therefore, as the voltage increases, the electrophoretic resolution actually decreases. In order to obtain the maximum resolution for separating DNA fragments by electrophoresis, the electric field strength should not be higher than 5V/cm.

The temperature of the electrophoresis system has no significant effect on the electrophoretic behavior of DNA in agarose gel. Generally, electrophoresis is carried out at room temperature. Only when the gel concentration is lower than 0.5%, can electrophoresis be carried out at 4 ℃ to increase the hardness of gel.

The research work of biochemistry and molecular biology often requires molecular hybridization of DNA separated by electrophoresis, but agarose is not suitable for hybridization operation. In 1975, Southren created a method of transferring DNA bands in situ to nitrocellulose membranes (NC membranes) for hybridization, which is called Southren blotting. 

Later, Alwine and others used similar methods for RNA imprinting, which is called Northern imprinting. In 1979, Towbin and others designed a device to transfer protein from gel to nitrocellulose membrane, transfer the protein to the membrane, and then react with corresponding antibodies and other ligands, which is called Western imprinting. This device makes the membrane, gel, filter paper, etc. into sandwich biscuits, and completes the transfer with low-voltage high current electrophoresis. In 1982, Reinhart et al. transferred the isoelectric focused protein zone from gel to a specific membrane by electric transfer method, called Eastern imprinting.

At present, there are various electrophoresis devices for nucleic acid and protein imprinting transfer available for sale both domestically and internationally, which enable fast and efficient imprinting transfer with good repeatability and wider applications. Polyacrylamide gel can also be used for imprint transfer electrophoresis, but when transferring proteins, gel cannot contain denaturants such as SDS and urea. There are also various options for supporting membranes used in transfer electrophoresis, with nylon membranes being more commonly used in recent years due to their good mechanical properties and resistance to baking and brittleness, making them more convenient to use than nitrocellulose membranes.

When performing imprinting transfer electrophoresis, it is important to note that the ion strength of the buffer should be low and the pH should be kept away from the pI, so that the protein carries more charge. Generally, a stable Tris buffer system is used. Also note that there can be bubbles between the gel and the support membrane. Appropriately increasing voltage or current can improve transfer speed, but it can also increase thermal effects. Therefore, voltage or the current should not be too high.

Generally, agarose gel electrophoresis can only separate DNA smaller than 20kb. This is because in agarose gel, when the effective diameter of DNA molecule exceeds the aperture of gel, under the action of electric field, DNA is forced to deform and squeeze through the sieve aperture, and straighten along the swimming direction, so the molecular size has little effect on mobility. If the direction of the electric field is changed in this way, the DNA molecule must change its conformation and straighten along a new swimming direction, and the turning time is closely related to the size of the DNA molecule. In 1983, Schwartz et al. designed a pulsed electric field gradient gel based on the characteristics of the elastic relaxation time of DNA molecules (extrapolated to the retention time of 0) and the size of DNA molecules. They alternately used two vertical non-uniform electric fields to make DNA molecules change direction in the gel, so that DNA is separated according to the size of the molecules. 

Later, Carle et al. improved the electrophoresis technique and discovered that periodic inversion of the electric field could also separate large molecule DNA through electrophoresis. The electrophoresis system is composed of a horizontal electrophoresis tank and two groups of independent and mutually perpendicular electrodes. One group of electrodes has N negative electrode and S positive electrode; the other group has W negative electrode and E positive electrode. A square agarose gel plate (10cm * 10cm or 20cm * 20cm) is placed in the center at 45 degrees. The electric field is alternately established between N-S and W-E. The duration of alternating changes in the electric field is related to the size of the DNA molecules to be separated. During electrophoresis, DNA molecules are in a continuous alternating electric field. First move towards the S pole, then change towards the E pole. 

Every time the direction of the electric field changes, DNA molecules need to have a certain amount of time to relax, change shape, and migrate. Only when the DNA molecule reaches a certain configuration can it continue forward. The net movement direction of DNA molecules is perpendicular to the loading line, allowing each component in the sample to form its own band along the same lane. Alternating pulse electrophoresis can effectively separate large molecular DNA with millions of base pairs. The angle and pulse time between the electrodes of newer instruments can be adjusted, making them more convenient to use.