RU2382081C2 - Method of separating and purifying nucleic acids - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: proposed method of separating and purifying DNA and RNA by centrifuging involves sorption of nucleic acids on a silicate sorbent - monodispersed spherical particles of silicon dioxide with size of 100-500 nm, with subsequent washing off impurities and elution.

EFFECT: method increases efficiency of separating nucleic acids.

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Description

The invention relates to the field of biotechnology, molecular biology and biochemistry and can be used in medicine. The invention is a method for the isolation and purification of nucleic acids (DNA and RNA) from biological and other samples.

Isolation and purification of nucleic acids are critical stages in molecular biological studies, including clinical diagnostic ones. There are various methods for the isolation and purification of nucleic acids, many of which are characterized by inconvenience of use, loss of nucleic acids and the duration of the procedures: centrifugation in a stepwise gradient of cesium chloride, column chromatography, extraction with phenol and chloroform, precipitation with ethanol and isopropanol [1].

Widespread methods for the isolation and purification of nucleic acids using silicate sorbents [2, 3], including using monodisperse and spherical particles of silicon dioxide with sizes from 0.05 to 10 microns [4]. They are based on the property of nucleic acids reversibly bind to the surface of silicate sorbents (fiberglass, glass powder) in solutions of salts and chaotropic agents. The sorbent with bound nucleic acids (DNA, RNA) is washed, as a rule, in alcohol solutions, after which the nucleic acids are removed from the sorbent in water or low-salt buffer. Moreover, various methods are known for using silicate sorbents for the isolation and purification of nucleic acids, for example, using liquid chromatography, as well as using magnetic particles with a silicate surface (5).

One of the common and convenient methods for the isolation and purification of nucleic acids is the simultaneous use of silicate sorbents and the centrifugation method in standard laboratory centrifuges. It was previously believed that for the isolation and purification of nucleic acids by centrifugation, it is most optimal to use silicate sorbents with an average particle size of more than 1 μm [6]. We have found that the use of spherical monodisperse particles of silicon dioxide can reduce the preferred average size of the sorbent used in the isolation and purification of nucleic frequencies using the centrifugation method to 100-500 nm.

Closest to the claimed invention (prototype) is a method for isolating nucleic acids using a silicate sorbent, described in [6]. The basis of this method for the isolation and purification of nucleic acids is the use of glass powder as a silicate sorbent - polydisperse particles of irregular shape with the most preferred size of 1-200 microns.

The disadvantage of the prototype is the low specific sorption capacity of the used sorbent (glass powder), since coarse micron-sized powder is used. Smaller powders having a large specific surface area are not recommended to be used according to the prototype, which is associated with poor redispersion of the resulting centrifugal sediments consisting of irregularly shaped polydisperse particles. The second disadvantage of the recommended material is that its preliminary processing is required, which consists in getting rid of particles of submicron sizes.

The objective of the invention was to reduce the amount of sorbent by weight used for the isolation and purification of nucleic acids by centrifugation.

The problem is solved in that in the proposed method, which includes adsorption of nucleic acids in buffers with high salt concentrations on a silicate sorbent, washing it from impurities and eluting nucleic acids in water or a low-salt buffer by centrifugation, monodisperse and spherical dioxide particles are used as the sorbent silicon with dimensions in the region of 100-500 nm, which increases the specific surface area of the sorbent and reduces the amount of sorbent used.

In the proposed process, nucleic acids are adsorbed on monodisperse spherical particles of silicon dioxide in a buffer solution containing a chaotropic agent. Monodispersed particles with nucleic acids are precipitated by high-speed centrifugation, washed from impurities, and then nucleic acids are eluted from the sorbent in water or in a low-salt buffer. As a result, the eluant is a solution of purified DNA or RNA, suitable for use in molecular biological research. The use of monodisperse spherical particles of silicon dioxide with a diameter of 100 to 500 nm provides both high sorption and short deposition time during centrifugation. Figure 1 shows the dependence of the specific surface area of spherical particles of silicon dioxide on their diameter. Figure 2 shows the dependence of the time of complete deposition of monodisperse spherical particles of silicon dioxide in a standard tube during centrifugation at 14,000 rpm of particles on their diameter.

The defining distinguishing features of the proposed method, in comparison with the prototype, are the following.

1. Use as a sorbent monodisperse particles of silicon dioxide.

2. Used monodisperse particles have a spherical shape, while the prototype uses polydisperse particles of irregular shape. The use of monodisperse particles of a spherical shape provides a good redispersion of a dense precipitate of particles formed by centrifugation associated with high molecular weight nucleic acids. In addition, the spherical shape of the particles provides a good desorption of nucleic acids in the final stage of obtaining purification.

3. The size of monodisperse particles of silicon dioxide is from 100 to 500 nm. Particles of this size have a large sorption surface. On the other hand, particles of such sizes maintain a high deposition rate in standard microcentrifuge tubes (up to 4 minutes) with centrifugation of 10000-16000 g.

4. The preferred size of monodisperse particles of silicon dioxide lies in the range of 400-500 nm, since particles of such sizes have a sufficiently high specific sorption of nucleic acids and at the same time a short time (up to 10 seconds) of complete deposition during high-speed centrifugation in standard microcentrifuge tubes.

Using the proposed method allows, in comparison with the prototype, to reduce the amount of sorbent by weight.

Example 1

Purification of high molecular weight DNA from human blood cells

For DNA extraction, archival blood cell samples fixed in solution (methanol: acetic acid in a ratio of 3: 1) were used. 10 μl of 0.5 M EDTA was added to 100 μl of the cell suspension, and the resulting mixture was lysed with 2 μl of proteinase K at 55 ° C for 10 minutes.

As the sorbent, we used monodisperse spherical particles of SiO ₂ in the form of an aqueous sol with a diameter of 300 ± 10 nanometers (Fig. 3) prepared at the JINR Laboratory of Nuclear Problems (Dubna, Moscow Region) in accordance with the previously described procedure [7]. Their size was 300 ± 10 nm, the concentration of 0.075 mg / μl.

From one sample for DNA purification, from 10 μl of the obtained cell lysate were added from 5 to 40 μl of a sorbent - sol of monodisperse particles of SiO ₂ . The sample volume was brought to 200 μl by adding a 6 M sodium iodide solution (pH 6.0), and then kept at room temperature for 10 minutes to bind genomic DNA to the carrier.

Particles with bound DNA were besieged by high-speed centrifugation at 14000 rpm (12000-16000 g) for 25 seconds at room temperature in a table centrifuge company "Eppendorf" (USA), model 5415C. The supernatant was removed, and the precipitate was resuspended in 1 ml of an 80% solution of ethanol in distilled water. Particles with bound DNA were again besieged by high speed centrifugation at 14,000 rpm for 25 seconds. The supernatant was removed and the precipitate was dried for 10 minutes. The pellet was resuspended in 40 μl of a 1 mM Tris-HCl solution (pH 8.5) and held for 10 minutes at 37 ° C to remove DNA from the carrier. The mixture was centrifuged at 14000 rpm for 10 seconds to precipitate particles, the supernatant with purified DNA was selected for analysis by standard gel electrophoresis.

Figure 4 - 1% agarose gel stained with ethidium bromide, with DNA purification results from 10 μl of human blood cell lysate: using 5 μl (lane 1), 10 μl (lane 2), 20 μl (lane 3) and 40 μl (lane 4) of the sorbent — an aqueous sol of monodisperse SiO ₂ particles with a diameter of 300 ± 10 nm; lanes 5 and 6 — control samples consisting of 10 and 5 μl, respectively, of a human cell lysate prior to purification; lanes 7-10 - sorbent precipitate remaining after removing DNA deposited in lanes 1-4, respectively.

In this example, DNA is isolated using a sorbent — monodispersed spherical particles of silicon dioxide with a size of 300 ± 10 nm, with a concentration of 0.075 mg / μl. The prototype describes the use of the same volume of sorbent - polydispersed particles of silicon dioxide with sizes of 1-200 μm, with a concentration of ~ 1 mg / μl. Thus, compared with the prototype, the amount of sorbent used was reduced by more than 10 times.

Example 2

Purification of high molecular weight DNA from human blood cells

DNA purification was carried out according to the method described in example 1. As a DNA sorbent, monodisperse spherical particles of SiO ₂ in the form of an aqueous sol with a size of 450 ± 10 nm were used (in contrast to Example 1, where particles with a size of 300 ± 10 nm were used). Particle deposition by high speed centrifugation in this example was carried out for 10 seconds (in contrast to 25 seconds in example 1).

Information sources

1. Methods of genetic engineering. Molecular Cloning: Per. from English / Maniatis T., Fritsch E., Sambrook J. - M.: Mir, 1984.

2. Boom R., Sol C.J.A., Salimans M.M.M., Jansen C. L., Wertheim-van Dillen P.M.E. and van der Noordaa J. // Journal of Clinical Microbiology. 1990. V.28. P. 495-503.

3. Vogelstein B. and Gillespie D. // Proc. Natl. Acad. Sci. USA 1979. V.76. P.615-619.

4. Patent US 2008/0241044 A1.

5. Patent US 5945525.

6. Patent US 5234809.

7. Bogush G., Tracy M. and Zukoski C. // J. of Non-Crystalline Solids. 1988. V. 104. P.95-106.

Claims (1)

The method of isolation and purification of nucleic acids by centrifugation, including their adsorption in buffers with high salt concentrations, washing from impurities and elution in water or low salt buffer using particles of silicon dioxide, characterized in that they use monodisperse spherical particles of silicon dioxide with sizes of 100-500 nm.

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