Molecular biology test method to explore two-agarose gel electrophoresis
Molecular biology test methods explore the characteristics of two-agarose gel electrophoresis technology, agarose gel. Natural agar (agar) is a polysaccharide, mainly composed of agarose (agarose, about 80%) and agarose (agaropectin). )composition. Agarose is a neutral substance composed of galactose and its derivatives, and has no charge. Agarose gel is a strong acid polysaccharide containing sulfate and carboxyl groups. Due to the charge of these groups, it can be produced under the action of electric field. Strong electroosmosis, combined with sulfate can affect the electrophoresis speed and separation effect with certain proteins. Therefore, at present, agarose is often used for electrophoresis support for plate electrophoresis, and the advantages thereof are as follows.
(1) Agarose gel electrophoresis is easy to operate, and the electrophoresis speed is fast. The sample can be electrophoresed without prior treatment.
(2) The structure of agarose gel is uniform and the water content is large (about 98%~99%). It is approximately free electrophoresis. The sample diffuses more free current and has little adsorption to the sample. Therefore, the electropherogram is clear, the resolution is high, and the repeatability is high. it is good.
(3) Agarose is transparent and UV-free, and the electrophoresis process and results can be directly detected and quantified by ultraviolet light.
(4) After electrophoresis, the zone is easy to stain, and the sample is easy to elute, which is convenient for quantitative determination. Made into a dry film can be stored for a long time.
At present, agarose is commonly used as an electrophoresis support to separate proteins and isozymes. The combination of agarose electrophoresis and immunochemistry develops into an immunoelectrophoresis technology, which can identify complex systems that cannot be identified by other methods. Due to the establishment of ultra-micro technology, 0.1 ug of protein can be detected.
Agarose gel electrophoresis is also commonly used to isolate and identify nucleic acids, such as DNA identification, DNA restriction endonuclease mapping, and the like. Because of the convenient operation, the simple equipment, the small sample amount and the high resolution, it has become one of the commonly used experimental methods in genetic engineering research.
Second, DNA agarose gel electrophoresis Separation of nucleic acids by agarose gel electrophoresis is mainly based on their relative molecular mass and molecular configuration, and also closely related to the concentration of gel.
1. Relationship between nucleic acid molecule size and agarose concentration (1) Size of DNA molecule In the gel, the migration distance (mobility) of the DNA fragment is inversely proportional to the logarithm of the base pair, so it is moved by a standard of known size. The distance of the unknown segment can be measured by comparing the distance of the unknown segment with the moving distance of the unknown segment. However, when the molecular size of the DNA exceeds 20 kb, it is difficult to separate them by ordinary agarose gel. At this time, the mobility of the electrophoresis is no longer dependent on the molecular size, and therefore, when the DNA is separated by agarose gel electrophoresis, the molecular size should not exceed this value.
(2) The concentration of agarose is shown in the following table. Different sizes of DNA need to be separated by electrophoresis using different concentrations of agarose gel.
Table agarose concentration and DNA separation range <br>Sepharose concentration/% 0.3 0.6 0.7 0.9 1.2 1.5 2.0
Linear DNA size / kb 60-5 20-1 10-0.8 7-0.5 6-0.4 4-0.2 3-0.1
2. Relationship between nucleic acid configuration and agarose gel electrophoresis The order of movement speed of different configuration DNAs is: covalently closed circular (cccDNA) > straight DNA> open-loop double-stranded circular DNA. When the agarose concentration is too high, the circular DNA (generally spherical) cannot enter the gel, and the relative mobility is 0 (Rm = 0), while the linear double-stranded DNA of the same size (rigid rod) can advance in the long axis direction. (Rm>0), it can be seen that the relative mobility of these three configurations mainly depends on the gel concentration, but at the same time, it is also affected by the current intensity, the buffer ionic strength and the like.
3. Electrophoresis method (1) Gel type Agarose gel electrophoresis for separating nucleic acids can be divided into vertical type and horizontal type (flat type). In horizontal electrophoresis, the gel plate is completely immersed in the electrode buffer for 1-2 mm, so it is also called a submersible type. At present, the latter is used more, because it is convenient to make glue and sample, the electrophoresis tank is simple, easy to manufacture, and it can be prepared according to the need to prepare gel plates of different specifications, which saves gel and is therefore popular.
(2) Buffer system In the absence of ions, the current is too small and the DNA migration is slow; on the contrary, the high ionic strength buffer generates a large amount of heat due to the large current, and in severe cases, the gel melts and the DNA denatures.
Commonly used electrophoresis buffers include EDTA (pH 8.0) and Tris-acetic acid (TEA), Tris-boric acid (TBE) or Tris-phosphoric acid (TPE), and the concentration is about 50 mmol/L (pH 7.5-7.8). The electrophoresis buffer is generally formulated as a concentrated stock solution and diluted to the desired multiple when used.
TAE has a low buffering capacity, and the latter two have a sufficiently high buffering capacity and are therefore more commonly used. Long-term storage of TBE concentrated solution will precipitate. To avoid this disadvantage, store 5× solution at room temperature and dilute 10 times 0.5× working solution to provide sufficient buffering capacity.
(3) Preparation of gel Use a diluted electrode buffer as a solvent, prepare a certain concentration of sol in a boiling Water Bath or a microwave oven, pour into a horizontal plastic frame or a vertical film, insert a comb, and naturally cool.
(4) Sample preparation and sampled DNA sample is dissolved in an appropriate amount of Tris-EDTA buffer containing 0.25% bromophenol blue or other indicator dye, containing 10%-15% sucrose or 5%~10% glycerol to increase Its specific gravity makes the sample concentrated. To avoid the possibility of sucrose or glycerol producing a U-shaped strip for electrophoresis, 2.5% Ficoll (polysucrose) can be used instead of sucrose or glycerol.
(5) Electrophoresis The experimental results of separation of 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 mobility of larger DNA fragments is relatively small. Therefore, as the voltage increases, the electrophoresis resolution decreases. In order to obtain the maximum resolution of the DNA fragment separated by electrophoresis, the electric field strength should not be higher than 5 V/cm.
The temperature of the electrophoresis system has no significant effect on the electrophoretic behavior of DNA in an agarose gel. Electrophoresis is usually carried out at room temperature, and only when the gel concentration is less than 0.5%, in order to increase the gel hardness, electrophoresis can be carried out at 4 °C.
(6) Dyeing and photographing Commonly used fluorescent dye ethidium bromide (EB) staining, observation of DNA bands under ultraviolet light, taking pictures with an ultraviolet analyzer, or outputting photos with a gel imaging system, and performing related data analysis.
3. Blot transfer electrophoresis Biochemistry and molecular biology research often requires molecular hybridization of electrophoretic DNA, but agarose is not suitable for hybridization. In 1975, Southren created a transfer of DNA regions in situ. A method of performing hybridization on a nitrocellulose membrane (NC membrane) is called a Southern blot method. Subsequently, Alwine et al. used a similar method for Northern blotting, dubbed the Northern blot. In 1979, Towbin et al designed a device for transferring proteins from a gel to a nitrocellulose membrane, transferring the protein to the membrane, and then the corresponding antibody. The ligand reaction, dubbed the Western blot, this device makes the membrane and gel, filter paper, etc. into a sandwich biscuit shape, and transfers with low voltage and high current electrophoresis. In 1982, Reinhart et al. used an electrotransfer method to transfer the isoelectrically focused protein band from the gel to a specific membrane, called the Eastern blot.
At present, there are a variety of electrophoresis devices for nucleic acid and Western blot transfer at home and abroad, which make the transfer speed of the blots high, high efficiency, good repeatability and wider application. Polyacrylamide gels can also be used for blot transfer electrophoresis, but when transferring proteins, the gel should not contain denaturing agents such as SDS and urea. There are also many options for the support film for transfer electrophoresis. In recent years, there are many nylon membranes because the nylon membrane has good mechanical properties, and the baking is not brittle, and it is more convenient to use than the nitrocellulose membrane.
When performing blot transfer electrophoresis, it is necessary to pay attention to the low ionic strength of the buffer, the pH should be far away from pI, and the protein has more charge, generally using a Tris-buffer system with better stability. Also note that there is a bubble between the gel and the support film. Properly increasing the voltage or current can increase the transfer speed, but it will also increase the thermal effect, so the voltage or current should not be too high.
Fourth, alternating pulse electric field gel electrophoresis
Generally, agarose gel electrophoresis can only separate DNA of less than 20 kb. This is because in an agarose gel, when the effective diameter of the DNA molecule exceeds the pore diameter of the gel, under the action of an electric field, the DNA is forced to deform through the sieve and straighten along the direction of the movement, so that the molecular size affects the mobility. Not big. When the direction of the electric field is changed in this way, the DNA molecule must change its conformation and straighten along the new migration, and the turn 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 elastic relaxation time of the DNA molecule (the extrapolated time of 0 is related to the size of the DNA molecule), alternating two orthogonal electric fields in the vertical direction to make the DNA The molecules constantly change direction in the gel, allowing the DNA to separate by molecular size. Later, Carle et al. improved the electrophoresis technique and found that the periodic inversion of the electric field also enables macromolecular DNA to be separated by electrophoresis. The electrophoresis system consists of a horizontal electrophoresis tank and two sets of independent, perpendicular electrodes. One set of electrodes has a negative electrode of N and a positive electrode of S; the other set of negative electrodes is W and the positive electrode is E. A square agarose gel plate (10 cm * 10 cm or 20 cm * 20 cm) was placed at a 45 degree center. The electric field is alternately established between NS and WE. The length of time that the electric field alternates is related to the size of the DNA molecule to be separated. When electrophoresed, the DNA molecules are in alternating alternating electric fields. First move to the S pole and then to the E pole. As each direction of the electric field changes, the DNA molecules will have some time to relax, changing shape and migration direction. Only when the DNA molecule reaches a certain configuration can it continue. The net movement direction of the DNA molecules is perpendicular to the sample line, so that the components in the sample form their respective zones along the same lane. Alternating pulse electrophoresis effectively separates millions of base pairs of macromolecular DNA. The angle between the newer instrument electrodes and the pulse time are adjustable, making it easier to use.
In addition, agarose plates are commonly used in immunoelectrophoresis techniques combined with immunoelectrophoresis techniques for a variety of immunoelectrophoresis.
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