Natural agar (agar) is a polysaccharide composed mainly of agarose (about 80%) and agaropectin. Agarose is composed of galactose and its derivatives of neutral substances, uncharged, while agar gum is a strong acidic polysaccharide containing sulfate and carboxylic acid groups, due to the charge of these groups, under the action of the electric field can produce a strong electroosmosis phenomenon, coupled with sulfate can be with certain proteins to affect the electrophoretic speed and separation effect. Therefore, at present, agarose is mostly used as electrophoretic support for plate electrophoresis, and its advantages are as follows.
(1) Agarose gel electrophoresis is easy to operate, the electrophoresis speed is fast, and the samples can be electrophoresed without prior treatment.
(2) The agarose gel has uniform structure and large water content (about 98%~99%), which is close to free electrophoresis, and the sample diffusion is more free-current, with minimal adsorption to the sample, so the electrophoresis pattern is clear, with high resolution and good repeatability.
(3) Agarose is transparent without ultraviolet absorption, and the electrophoresis process and results can be directly detected and quantitatively determined by ultraviolet light.
(4) The zones after electrophoresis are easy to be stained, and the sample is very easy to be eluted, which is convenient for quantitative determination. The dry film can be preserved for a long time.
At present, agarose is commonly used as electrophoretic support to separate proteins and isoenzymes. The combination of agarose electrophoresis and immunochemistry has led to the development of immunoelectrophoresis technology, which is capable of identifying complex systems that cannot be identified by other methods, and due to the establishment of the ultra-micro technology, 0.1ug of protein can be detected.
Agarose gel electrophoresis is also commonly used in the separation and identification of nucleic acids, such as DNA identification, DNA restriction endonuclease mapping. Because this method is easy to operate, the equipment is simple, the amount of samples required is small, and the resolution ability is high, it 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 mass and molecular configuration, and is also closely related to the concentration of the gel.
1. The relationship between nucleic acid molecular size and agarose concentration
(1) the size of the DNA molecule in the gel, the DNA fragment migration distance (mobility) and the logarithm of the base pairs is inversely proportional to the size of the unknown fragment can be measured through the known size of the distance of movement of the standard and the unknown fragment of the distance of the movement of the comparison, the size of the unknown fragment. However, when the size of the DNA molecules exceeds 20 kb, it is difficult to separate them on an ordinary agarose gel. 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.
(2) Concentration of agarose As shown in the table below, different sizes of DNA need to be separated by electrophoresis with different concentrations of agarose gel.
Table Agarose concentration and DNA separation range
Agarose concentration /% 0.3 0.6 0.7 0.9 1.2 1.5 2.0
Nematic DNA size/kb60-520-110-0.87-0.56-0.44-0.23-0.1
2. Relationship between nucleic acid conformation and separation by electrophoresis on agarose gels
The order of moving speed of different conformations of DNA is as follows: valence-supplied closed circular DNA (covalently closed circular, cccDNA)> straight-line DNA> open circular double-stranded cyclic DNA. when the concentration of agarose is too high, the cyclic DNA (generally spherical) can not enter into the gel, and the relative mobility is 0 (Rm=0), while the same size of straight-line DNA has the same relative mobility, and the relative mobility is 0 (Rm=0). 0), while linear double-stranded DNA of the same size (rigid rod) can advance in the direction of the long axis (Rm>0), which shows that the relative mobility of these three configurations depends mainly on the gel concentration, but also, it is affected by the current strength, buffer ionic strength, etc.
3. Electrophoresis methods
(1) Gel type
Agarose gel electrophoresis for separating nucleic acids can be divided into vertical type and horizontal type (plate type). In horizontal type electrophoresis, the gel plate is completely immersed in the electrode buffer under 1-2mm, so it is also called diving type. At present, the latter is more often used, because it is more convenient to make gel and add samples, electrophoresis tank is simple, easy to make, and can be prepared according to the need for different specifications of the gel plate, saving gel, and therefore more popular.
(2) Buffer system
When there is a lack of ions, the current is too small, and the DNA migration is slow; on the contrary, the buffer with high ionic strength will produce a lot of heat due to the current is too large, which, in serious cases, will cause melting of the gel and denaturation of DNA.
Commonly used electrophoresis buffers are EDTA (pH 8.0) and Tris-acetic acid (TAE), Tris-boronic acid (TBE) or Tris-phosphoric acid (TPE), etc., with a concentration of about 50 mmol/L (pH 7.5~7.8). The electrophoresis buffer is usually prepared as a concentrated reservoir solution and diluted to the required multiplicity when it is ready for use.
TAE buffering capacity is low, the latter two have high enough buffering capacity, so more commonly used.TBE concentrated solution for long-term storage will be precipitated, in order to avoid this drawback, room temperature storage 5 × solution, when used to dilute 10 times 0.5 × working solution that can provide sufficient buffering capacity.
(3) Preparation of gel
The diluted electrode buffer as a solvent, using a boiling water bath or microwave oven to prepare a certain concentration of sol, poured into the horizontal gel frame or vertical gel film, inserted into the comb, and cooled naturally.
(4) Sample preparation and spiking
DNA samples were dissolved in appropriate amount of Tris-EDTA buffer, which contained 0.25% bromophenol blue or other indicator dyes, 10%-15% sucrose or 5%-10% glycerol to increase its specific gravity and to concentrate the sample. To avoid the possibility that sucrose or glycerol may produce U-shaped bands in the electrophoresis results, 2.5% Ficoll (polysucrose) can be used instead of sucrose or glycerol.
(5) Electrophoresis
The results of the experimental conditions of agarose gel separation of macromolecular DNA showed that the separation effect was better at low concentration and low voltage. Under low voltage conditions, the electrophoretic mobility of linear DNA molecules was proportional to the voltage used. However, the increase in mobility of larger DNA fragments was relatively small at increasing electric field strength. Therefore, as the voltage increases, the electrophoretic resolution decreases instead, and in order to obtain the maximum resolution of electrophoretically separated DNA fragments, the electric field strength should not be higher than 5 V/cm.
The temperature of the electrophoresis system does not have a significant effect on the electrophoretic behavior of DNA in agarose gels. Electrophoresis is usually carried out at room temperature, and only when the gel concentration is less than 0.5%, electrophoresis can be carried out at 4°C in order to increase the gel hardness.
(6) Staining and photographing
The fluorescent dye ethidium bromide (EB) is commonly used for staining, observing the DNA bands under ultraviolet light, photographing them with a UV analyzer, or outputting the photographs with a gel imaging system, and analyzing the relevant data. Research work in biochemistry and molecular biology often requires molecular hybridization of electrophoretically separated DNA, but agarose is not suitable for hybridization operations. In 1975, Southren created a method of in situ transferring DNA bands onto nitrocellulose-based membranes (NC membranes), followed by hybridization, which is known as the Southren blotting method. Subsequently, Alwine et al. used a similar method for RNA blotting, which was jokingly called Northern blotting, and in 1979, Towbin et al. designed a device to transfer proteins from a gel to a nitrocellulose membrane, transferring the proteins to the membrane and then reacting them with ligands such as the corresponding antibodies, which was jokingly called Western blotting, and this device made the membrane and the gel and filter paper into the shape of a sandwich cookie In 1982, Reinhart et al. used the electrotransfer method to transfer the protein bands after isoelectric focusing from the gel to the specific membrane, called Eastern blot.
At present, there are many kinds of electrophoresis devices for nucleic acid and protein blotting transfer sold at home and abroad, which make the blotting transfer fast and efficient, reproducible and more widely used. Polyacrylamide gel can also be used for blot transfer electrophoresis, but when transferring proteins, the gel must not contain SDS, urea and other denaturants. Support membrane for transfer electrophoresis also has a variety of choices, in recent years with nylon membrane more, because nylon membrane mechanical properties, baking is not brittle, the use of more convenient than nitrocellulose membrane.
To carry out the blotting transfer electrophoresis, it should be noted that the ionic strength of the buffer should be low, the pH should be away from the pI, so that the protein with a higher charge, generally with a better stability of the Tris-buffer system. Also note that there can be air bubbles between the gel and the supporting membrane. Appropriate increase in voltage or current can improve the transfer rate, but will also increase the thermal effect, so the voltage or current should not be too high. General agarose gel electrophoresis can only be separated less than 20kb of DNA, this is because in the agarose gel, the effective diameter of the DNA molecule more than the pore size of the gel, under the action of the electric field, forcing the DNA deformation squeezed through the sieve holes, and along the direction of the straightening of the swim, and thus the molecular size of the mobility of the impact is not great. If this time to change the direction of the electric field, the DNA molecule must change its conformation, along the new swimming direction straightening, and the steering time and the size of the DNA molecule is extremely close relationship. 1983 Schwartz et al. according to the elastic relaxation time of DNA molecules (extrapolated to the 0 retention time) and the size of DNA molecules related to the characteristics of the design of the pulsed electric field gradient gel, alternating between two perpendicular direction of the Uneven electric field, so that the DNA molecules in the gel constantly change direction, so that the DNA according to the molecular size separation. Later, Carle and others improved the electrophoresis technique and found that periodic inversion of the electric field could also separate large DNA molecules by electrophoresis. The electrophoresis system consists of a horizontal electrophoresis tank and two sets of independent, perpendicular electrodes, one set of electrodes with negative electrode N, positive pole S; the other set of electrodes with negative electrode W, positive pole E. A square agarose gel plate (10cm*10cm or 20cm*20cm) is placed in the center at 45 degrees. The electric field was established alternately between N-S and W-E. The length of time for which the electric field is alternately changed is related to the size of the DNA molecule to be isolated.
During electrophoresis, the DNA molecules are in an electric field that alternates at successive intervals. It first moves toward the S pole and then changes to the E pole. At each change in the direction of the electric field, the DNA molecules have to have a certain amount of time to relax and change shape and migration direction. Only when the DNA molecule reaches a certain configuration, it can continue before the time. the net direction of movement of DNA molecules is perpendicular to the sampling line, so that the components of the sample along the same lane to form their own zones. Alternating pulse electrophoresis can effectively separate large DNA molecules with millions of base pairs, and the angle between the electrodes and the pulse time of the newer instruments are adjustable, making them more convenient to use.
In addition, agarose plates are commonly used for a variety of immunoelectrophoresis techniques that combine immunodiffusion techniques with electrophoresis.