Shanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai 20031Yu Xingming
First of all, a brief introduction.
In recent years, the separation and purification of plasmid DNA is represented by the separation from Escherichia coli. In view of the important position of Escherichia coli in molecular biology research, the separation and purification of quality control DNA (plasmid DNA) from Escherichia coli has become an important topic in ultracentrifugation technology. The rapid separation and purification of plasmid DNA put forward higher requirements for ultracentrifuge equipment (ultracentrifuge, rotor and auxiliary equipment).
Escherichia coli is a typical prokaryotic cell organism. Because prokaryotic cells lack the inner membrane system of their nuclei, which can separate various functional components into specialized and locally independent regions, it has no organelles (nucleus, endoplasmic reticulum, Golgi apparatus, filaments, lysosomes, etc.). ) is contained in its nucleus. Electron microscope micrographs show that Escherichia coli has two distinguishable internal regions-cytoplasm and nucleoplasm, which are surrounded by a thin plasma membrane and a thick cell wall, and some flagella are attached to the outside of the cell wall. Plasmid DNA is located in the nuclear region and exists in filament form. In many cases, this filament is a concentrated product of some fragments of extremely long circular DNA folded.
According to the microstructure of Escherichia coli, the pretreatment sequence before ultracentrifugation purification of plasmid DNA is as follows:
Escherichia coli → removing cell wall with lysozyme → using surfactant such as SDS, Trit X- 100 and other EE cell membranes → precipitating most DNA, RNA and protein (more than 90%) with acetic acid tray.
After adding te buffer (10 m-mtris-HCl LMMEEDTA, pH 8.0), the precipitate can be deproteinized and RNA can be removed on the molecular sieve. You can also use ultracentrifugation to remove protein, RNA, fractionated DNA or DNA fragments. This paper will summarize the new progress of the latter method in recent years.
Second, the latest progress in the separation of plasmid DNA by ultracentrifugation
1. Traditional separation method: A few years ago, due to the limitation of equipment conditions, the plasmid DNA was generally separated by CsCl balanced isodensity centrifugation, and the gradient was formed by itself. Taking the capacity of a single tube 10~ 12ml as an example, the separation with a flat rotor is 36.000rpm×60 hours, and the separation with an angular rotor is 45 × 36 hours. The former includes acceleration and deceleration, which requires a driving life of1.300 million revolutions, while the latter also requires 65438+. Considering that the total life of the ultracentrifuge at that time was 654.38+000 ~ 20 billion revolutions, there is no doubt that the cost of each experiment is too high, and the large amount of CsCl and high price make this separation and purification work very expensive.
2. New progress:
Centrifugal separation (1) overspeed riser head (made of Qin alloy or carbon fiber): Since 1975 riser head went to the world, in recent years, the riser head developed by major centrifuge manufacturers has a single tube capacity of 0.2ml to 4Oml, and the maximum rotation speed ranges from 50,000rpm to120,000 rpm, and the max can reach.
700,000 XG, a new model and rotor developed in 1990s, can make the experiment of vertical tube centrifugal separation of plasmid DNA handy.
The main points of separating and purifying plasmid DNA by vertical tube head are as follows:
Because of the shortest settling distance, the shortest centrifugation time, high efficiency and low consumption;
The longitudinal cross-sectional area of the centrifugal tube is larger than the transverse cross-sectional area of the centrifugal tube, and the capacity of the pure product area is larger during centrifugal sedimentation, and the separation purity is high and the sample loading is also large without precipitation attachment or very close precipitation attachment; Hydrostatic pressure will not damage biological particles because of excessive hydrostatic pressure. The hydrostatic pressure of the sample particles in the centrifuge tube is:
ω: angular velocity
R: the distance between the position of the sample particle and the center of rotation (in centimeters)
R 1: The minimum centrifugal radius of the rotor is rmin (cm), which is the distance between the liquid level and the rotation center when using gmax for centrifugation.
Pa: initial density (minimum density of prefabricated gradient and minimum density at the end of autogenous gradient centrifugation).
A: The gradient of step skin.
In ultracentrifugation, the value of P is quite large, and it is generally believed that P≤ 1500kg/cm2 will not damage biological particles. The vertical pipe head (r-r 1) is very small, and relatively speaking, P is also quite small (102 order of magnitude). However, it is often necessary to check the P value before centrifugation. If it is too large, RNA precipitates are attached to the wall of the centrifuge tube during the separation and purification of plasmid DNA through the vertical tube. When deceleration and gradient direction change, plasmid DNA region "eats grass" from precipitation region, which makes a small amount of RNA mixed into DNA and affects purity.
When centrifugation is carried out at a very high speed (for example, over 80,000 rpm), this effect is small because RNA adheres tightly to the wall.
(2) Centrifugal separation of near-vertical tube heads: In order to eliminate the pollution of RNA precipitation formed on the tube wall when the vertical tube heads centrifuge plasmid DNA, and at the same time, to improve the shortcoming that the general inclined tube heads (with an inclination angle of 25 ~ 35) have a long settling distance, so the separation time is also long, a variety of near-vertical tube heads (that is, near-vertical tube rot, The angle between the central axis of the longitudinal section of the centrifuge tube and the driving axis of the centrifuge is 7.5 ~ 10, and the rotation speed ranges from 65,000 rpm ~ 120, and the maximum rotation speed can reach 646000×g ..NVT The development of NVT (or nt) rotor is mainly designed for the separation of plasmid DNA. It is also suitable for the separation and purification of mitochondrial DNA, chromosomal DNA, RNA and serum lipoprotein.
The characteristics of using NVT(NT) to separate and purify quality control DNA are as follows:
The time required for centrifugation is slightly longer than that of vertical tube rotor (increased by 30%~40%), but shorter than that of general inclined rotor (60%~70% of the separation time of similar inclined rotor).
Although the composition of the outer wall of RNA centrifuge tube is small because of the small inclination angle, after adding surfactant (such as 0.1%~ 0.001%trimnx-100), RNA precipitation can quickly slide to the bottom of centrifuge tube (that is, RNA has reached the bottom when gradient is formed), and the purity of cytoplasmic DNA is not affected during gradient transformation.
Small hydrostatic pressure, running at very high speed will not damage biological particles;
The longitudinal section of centrifugal tube is larger than that of general angular and rotary head, with high separation purity and large sample loading.
(3) Discontinuous gradient separation: The traditional method of DNA separation and purification for quality control is gradient equilibrium isodensity centrifugation with gold tube CsCl. At the beginning of centrifugation, the CsCl density of the gold tube is uniform and the samples are evenly distributed in it.
The centrifugation time of CsCl self-forming gradient can be calculated by the following formula:
Where n: actual rotational speed (rpm)
Floating density of samples (such as plasmid DNA) in CsCl.
R: distance from the center of rotation to the center of pure sample belt (cm). As a preliminary solution, plasmid DNA is centrifuged, and the R position can be located in the middle of the centrifuge tube, and the average publishing point of R is-10%. (rmax-rmin).
F: The constant coefficient depends on the initial density of gradient materials and solutions (Table 1).
S20.w: The sedimentation coefficient of the sample (plasmid DNA) in water at 20℃ can be determined by referring to relevant literature.
[Note] TCA is trichloroacetic acid and TFA is trifluoroacetic acid in the table.
The centrifugal time calculated by the above formula is very close to the actual centrifugal result. It can be seen from the formula that increasing the T- inverse ratio (N4, r2) will obviously reduce the centrifugation time, especially the effect of increasing the rotation speed, on the premise that CsCi does not crystallize and precipitate. However, for some rotating heads with low rotating speed (such as below 65,000rpm), it takes too long for CsCl to form its own gradient (10 hour), so it takes a long time to form the pure sample area of plasmid DNA.
As an improvement of the initial equidensity condition of the whole tube, in recent years, the balanced equidensity centrifugation of stepped discontinuous CsCl gradient has been developed, that is, the initial gradient of the centrifugal tube is divided into two parts:
Lower part: high density area (p =1.80-1.81g/cm3), accounting for 1/3 of the total capacity.
Upper part: low density area (p= 1.46- 1.48g/cm), accounting for 2/3 of the total capacity. The experiment shows that the second-order discontinuous CsCl gradient takes much less time than the whole tube isodensity centrifugation (taking 12ml vertical tube rotor as an example, the gradient of plasmid DNA and CsCl is 55,000rPm. Discontinuous two-step centrifugation is 5.5.
Hours, the whole tube isodensity centrifugation is 8 hours).
Second-order discontinuous gradient centrifugation, the sample is added to the high-density area near the bottom of the centrifugal tube, and its R is very large for any kind of rotor; Moreover, in the low density region (low centrifugal force port velocity region), only the sample floats without the sample settling; Close to DNA floating
The initial density difference in density area is large, the gradient forms itself and the time is short; Due to the above reasons, the centrifugation time is shortened. (4) Ultra-high-speed multi-stage (rotational speed) separation: Obviously, increasing the rotational speed according to the T formula will accelerate the formation of CsCl gradient and shorten the centrifugation time accordingly. However, at a certain temperature and high speed, long-term centrifugal separation is likely to produce local crystallization and precipitation of high-density CsCl, and local precipitation will not only affect the gradient, but also lead to centrifugal failure or accident due to the damage of the centrifugal tube wall of solid CsCl. Therefore, for the centrifugal separation experiment of plasmid DNA, due to the programmable operation characteristics of the new overspeed machine, it can gradually move from extremely high speed to slightly lower speed, and each experiment is divided into multiple speed gears, which not only improves the efficiency, but also ensures the success and safety of the experiment.
Of course, this kind of experiment has to be explored many times before the commonly used centrifugal scheme can be formed. Beckman Company of the United States and Hitact 1i ko ki Co., Ltd. of Japan have done a lot of experiments on their own advanced overspeed machines, and recommended reliable and efficient centrifugal psychological sequence [2,31.
Iii. Examples of ultracentrifugation separation of typical plasmid DXA
1. Examples of traditional separation:
Example: (1) single pipe capacity 12rnl, angular rotor with maximum speed below 50,000rpm, 12PA sealed pipe, and new overspeed in each factory.
Sample +TE solution (prepared to pH8.0), EB dosage 0.2mg/ural, CsCl added to make the whole tube p =1.57g/ml 45,000 rpm× 36 hours, at 20℃, slow down or 55,000 rpm×16 hours+40,000 rpm.
Features: The separation effect is good, but the sample width is wide, the separation time is long, and the life of transmission parts is nearly 65.438+0 billion revolutions.
Example (2) An iron hanging chair with a single tube capacity of 5ml and a maximum rotating speed of 65,000rpm, with a flat rotating head and 5PA tubes, and the sample configuration is the same as the example (1).
Features: The same example (5) (it takes 20 million revolutions to drive life).
36,000 rpm× 55 hours, 20℃ or 32,000 rpm× 70 hours, 20℃
Separation results and characteristics: the separation results are ideal, the plasmid DNA band is very narrow, and RNA settles at the bottom of centrifuge tube. However, the centrifugation time is too long and the cost is high (it takes 1. 1 100 million ~1.300 million revolutions to drive the service life).
2. Centrifugal separation of vertical tube head;
Example (3) Hitachi cp 100α mainframe, P 100VT rotor (700000×g, 8×5ml), and 5PA sealed tube, with the same sample and gradient configuration (1).
100000 rpm × 1 hour 50 minutes, 20℃, deceleration (7th gear).
Features: The centrifugal result is similar to the example (1). Due to the extremely high rotation speed, RNA precipitates on the wall very tightly, and there is little pollution to DNA during deceleration. The centrifugation time is short, the efficiency is high, and the cost is low (the driving life needs 0.10.2 billion revolutions).
Example (4) Qin riser faucet, the maximum speed is 50000 rpm, the capacity is 8×40ml, and the sealing tube is 40PA. The sample car configuration is as follows: < 1 > 50000 rpm× 24 hours, 20℃, slow deceleration.
Features: large-capacity separation, RNA precipitation slightly contaminated DNA bands.
3. Nearly vertical tube head separation
Example (5)Beclunar 1NVT90 is switched to XL-90 mainframe, when it is 8×5, the tube is sealed with 5PA, and the maximum speed is 90000 rpm, 646,000× g g ... sample +TE solution (pH8.0), the amount of E B is 0.2mg/mh, and tritmx-/kloc. 78,000 rpm× 4 hours, 20℃, deceleration.
Features: Due to the function of Triton X- 100, RNA precipitation slides to the bottom of centrifuge tube at an accelerated speed, and there is no pollution to DNA during deceleration. The separation result is ideal (it takes 20 million revolutions to drive the unit).
Example (6) Hitachi CS 12OEX or Beckman.
TLX micro-overspeed machine, the maximum speed 120, the NVT(NT) rotor of OO RPM is 8×2, and the configuration of sealing tube, sample and gradient solution is basically the same as that of Example (5), but the configuration of CsCL is P = 1.55 z/ml,120000 RPR.
4. Discontinuous step gradient separation;
Example (7) Japan Hitachi SRP83VT rotary head, 80,000rprm, 549,000g, 8× 5,5pa sealed tube (Hitachi CPα, β series machine or SC Xia series machine), gradient liquid configuration:
TE solution +Cscl, p= 1.47g/, ***3.5ml, is first injected into a 5PA sealed tube.
TE solution+sample +CsCl is prepared into p =1.81g/mhe.b.0.2mg/ml, and * *1.5ml is slowly injected into the bottom of the tube with a syringe to float the original p= 1.47 solution.
83, ooorpm× 1 hour, 20℃, slow acceleration, slow deceleration, the result is the same as in Example (3).
Features: The second-order discontinuous gradient is adopted, which shortens the time for CsCl to form its own gradient. Ultra-high speed, compact RNA precipitation, little pollution to DNA bands, high efficiency and low cost (only using 50 million rpm to drive the unit life). The disadvantage is that the sample addition is slightly small.
Example < 8 > Japanese Hitachi RP55VF: when rotating the head, the speed is 55000 rpm, 293,000× g, 12×5, and the configuration of the sample and gradient liquid is the same as in Example (7), 55000 rpm×4.5 hours, 20°c, slow acceleration and deceleration. The result is similar to that in Example 7.
5. Ultra-high speed multistage (speed) separation:
Example (9): 9): Beckman XL-90 overspeed machine, 90,000 rpm NVT-90 rotary head, 645,000xg, 8×5ml, 5. 1PA sealing tube, and the sample and gradient liquid configuration are the same as in Example (5).
Multi-stage separation: 90,000rpm× l.5 hours+87,000 rpm× 0.25 hours+83,000 rpm× 0.25 hours +8 1000 rpm×0.50 hours +8 1000 rpm×0.50 hours, at 20℃, slow down. (The above experiments can also be done on the Japanese OMI CP 100α or 90α mainframe, and the results are the same).
Features: The result is the same as in Example (5), but the time is reduced to 3 hours (0.10.6 billion rpm).
Example: (10)Hitact 1i micro overspeed CS- 120EX or CS- 10OEX micro overspeed, SLOAT 5 corner, 100, OORPM, 550,000× g, 8×5ml 100, OORPM× 4.5 hours ++98, OORPM×15+05+96. OORPM× 30 ++94, OORPM× 30 ++90, OOOrpm×25 ++85. OOOrpmX30 points (* * 7 hours
Features: Using angle rotor and micro-overspeed machine, under the condition of small centrifugal force (compared with large overspeed machine), the centrifugation time is shorter, RNA sinks to the bottom of the tube, and the separation result is ideal.
Four. Matters needing attention in ultracentrifugation separation of plasmid DNA
1. Pollution Prevention and Dehydrogenase Pollution Prevention: Dehydrogenase can degrade or denature DNA, and it is everywhere (on the skin, on the surface of instruments that have not been cleaned and disinfected ...), so we should pay attention to this point in every step of plasmid DNA separation experiment. An effective method is to disinfect instruments (centrifuge tubes, lids, syringes, pipettes, containers, etc.). Although we can add DFP (diisopropyl fluoride) or PMSF (benzophenone atmosphere) to inhibit the degradation and use of dehydrogenase, the main means is cleaning and disinfection. In order to prevent ribosomes from polluting the skin, surgical gloves should be worn during surgery.
2. Prevent heavy metal ions in heavy metal salts from combining with DNA to form a complex: CsCl is an ionization medium. Before centrifugation, CsCl should pass through a column to eliminate Cs ions, and the utensils in contact with the sample should also be cleaned with deionized water.
3. The sample loading is related to the sample concentration. In order to improve the resolution, the amount of samples should be controlled, and the appropriate amount should be determined according to previous experiments. The sample size per ml of gradient solution is about10 μ z ~ 20 μ g z ~ 20 μ g.
4. For angular, vertical and nearly vertical rotating heads, self-forming gradient balance and equal density separation can be provided. 0~ 100Orpm is fast acceleration, 1, 000rprn~0 ~ 0 is slow deceleration, and the step gradient requires slow acceleration and slow deceleration. In modern times, this machine has provided a good reference example for users to choose the acceleration and deceleration rate.
5. The temperature of the rotor at the beginning of centrifugation should be basically the same as the working temperature during centrifugation (5℃ in soil), and the precooling temperature of the rotor is too low (lower than 10oC). When the rotor is centrifuged at a very high speed for a short time, CsCl may precipitate because it is too late to heat up, which may lead to experimental failure or accidents.
6. Appropriate sampling method: Plasmid DNA is easy to be separated by ultracentrifugation, and the sampling method and operation level after centrifugation will determine the recovery rate and sample purity. First, the sampling environment should be close to the centrifugal working temperature (5℃); There should be no vibration source on the experimental platform when sampling; According to the laboratory conditions, transverse puncture, empty hole at the bottom, eccentric cutting tube or gradiometer are selected accordingly. However, the department must be careful.
7. Suitable pH value. During the whole experiment, the fake nucleic acid should be kept in TE solution with pH8.0. Too high or too low pH value can also denature DNA.
8. Volume of centrifuge tube and added liquid: Because the rotating speed used in modern ultracentrifugation to separate cytoplasmic DNA is very high, the material requirements of 7-core tube are also very high. Generally, the centrifuge tube used in this kind of experiment is only used once. It is best to use a sealed tube, and it is best to choose a PA tube for the first section. Centrifugal tubes shall not be stored for more than 2~3 years, and samples using sealed tubes or thin-walled tubes with covers shall be filled up. Use the spinner to level the nozzle of EE for 2 ~ 3mm to prevent the meniscus from overflowing or the nozzle from overturning.