RU2495925C2 - Method of separating and purifying nucleic acids from liquid medium (versions) and plastic vessel for sorbing nucleic acids from liquid medium - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: disclosed is a plastic vessel for sorbing nucleic acids from a liquid medium. The inner surface of the vessel is coated with silicon oxide via thin-film synthesis, which involves ion-plasma sputtering carried out in an ultrahigh vacuum by sputtering a silicon oxide target with a stream of Ar+ ions. The thickness of said coating is in the range of 2-400 nm. Also disclosed are methods of separating and purifying nucleic acids from a liquid medium (versions) using said vessel. First, nucleic acids are sorbed on inner walls of the vessel and impurities are then washed off. After adding an eluting solution, the vessel is heated to 95°C to separate DNA or to 65°C to separate RNA, intensely shaken and a solution of nucleic acids is collected in another vessel.

EFFECT: invention improves sorption properties of the vessel, provides uniformity of the coating on the entire surface of the vessel, on both a large area and a limited area, high optical transparency of the vessel, fewer operations for separating and purifying nucleic acids.

15 cl, 1 dwg

Description

The invention relates to the field of biotechnology, in particular to a method for the isolation and purification of nucleic acids.

The invention is known "METHOD FOR ISOLATION AND PURIFICATION OF NUCLEIC ACIDS" (Patent RU No. 2382081, publ. 02.20.2010), which proposes a method for the isolation and purification of DNA and RNA by centrifugation, providing for the sorption of nucleic acids on a silicate sorbent - monodisperse spherical silicon dioxide particles 100-500 nm in size - with subsequent washing from impurities and elution. The method allows to increase the efficiency of the allocation of nucleic acids.

The disadvantage of this invention is the low degree of uniformity and uniformity of the coating, as well as its corresponding productivity and manufacturability. High degree of deformation of the base material. Particles are not located on the entire surface of the vessel.

The invention is known "Device for DNA isolation and method" (Patent WO 2004046231 (A1), publ. 2004-06-03), in which according to the first embodiment of the invention, a vessel made of plastic for sorption of nucleic acids from a liquid medium includes a coating of silicon oxide on the inside the surface of the vessel made of plastic having a monolayer of particles, at least on part of the surface of the vessel, the particles are selected from quartz, quartz gel, or glass. A method for isolating DNA from a crude base includes sorption on the inner walls of the vessel, washing from impurities, and elution in saline.

The disadvantage of this invention is the low productivity and manufacturability, low sorption properties, the impossibility of uniform coating over the entire surface of the vessel, and deformation of the base material, low optical transparency is possible both on a large area and in a limited area.

This invention is the closest technical solution to the claimed solution, i.e. prototype.

The objective of the proposed solution is to increase the productivity and manufacturability of the process of isolation and purification of nucleic acids, increase the speed of the process, reduce the number of operations performed, increase the purity of the obtained nucleic acids, increase the sorption properties of the vessel in which the process is performed, ensure uniform coverage over the entire surface of the vessel, and how over a large area and in a limited area, increasing the optical transparency of the vessel.

The problem is solved by creating a plastic vessel for sorption of nucleic acids from a liquid medium, including a coating of silicon oxide on the inner surface of the vessel, which is a nanocoating (defined as a coating with a thickness of 1 to 100 nm) made by thin-film synthesis.

In addition, the plastic can be selected from the group comprising polyvinyl chloride, polyethylene, polypropylene, polystyrene, polycarbonates, acrylonitrile butadiene styrene, polyurethanes, polyamides, thermoplastic elastomers, polysulfones and polyether ether ketones, as well as mixtures thereof.

In addition, the vessel can be made optically transparent.

In addition, the vessel may be made in the form of a test tube.

In addition, the specified thin-film synthesis can be implemented at ultrahigh vacuum.

In addition, the specified thin-film synthesis may include ion-plasma spraying.

In addition, ion-plasma sputtering may include sputtering a silicon oxide target with a stream of Ar + ions in a vacuum.

In addition, the inner surface of the vessel before spraying can be chemically cleaned with alcohol in an ultrasonic bath.

In addition, the thickness of the nanocoating does not exceed 25 nm.

The problem is also solved by creating a method for the isolation and purification of nucleic acids from a liquid medium, including their sorption on the inner walls of the vessel, washing from impurities and elution in saline solution, using silicon oxide nanocoating made by thin-film synthesis as a sorbent.

In addition, a silicon oxide nanocoating with sizes from 1 to 25 nm is used.

In addition, a chaotropic reagent, a pH buffer are additionally added to the vessel with thin-film spraying of SiO ₂ , the mixture is shaken, then poured or taken by automatic pipette, alcohol is added to the vessel in a volume equal to the nominal volume of the vessel, shaken and drained.

In addition, after adding the eluting solution, the vessel is heated to 95 ° C to isolate DNA or to 65 ° C to isolate RNA, shake vigorously, and the nucleic acid solution is taken to another container.

Also, according to option 2, the problem is solved by creating a method for the isolation and purification of nucleic acids from a liquid medium, including their sorption on the inner walls of the vessel, washing from impurities and elution in saline solution, using SiO ₂ nanocoating made by thin-film synthesis as a sorbent in a vessel, at the same time a chaotropic reagent, a pH buffer are added to the vessel, the mixture is shaken, then drained or removed by an automatic pipette, then alcohol is added to the vessel, equal in volume, equal to the nominal value Nome volume of the vessel, shaken and decanted.

In addition, a silicon oxide nanocoating with sizes from 1 to 25 nm is used.

In addition, after adding the eluting solution, the vessel is heated to 95 ° C to isolate DNA or to 65 ° C to isolate RNA, shake vigorously, and the nucleic acid solution is taken to another container.

Also, according to option 3, the problem is solved by creating a method for the isolation and purification of nucleic acids from a liquid medium, including their sorption on the inner walls of the vessel, washing from impurities and elution in saline, heating the vessel, using SiO ₂ nanocoating made as a sorbent, made by thin-film synthesis in a vessel, while a chaotropic reagent, pH buffer is added to the vessel, after adding an eluting solution, the vessel is heated to 95 ° C to isolate DNA or to 65 ° C to isolate RNA, intens shake vigorously and take the nucleic acid solution into another container.

In addition, a silicon oxide nanocoating with sizes from 1 to 25 nm is used.

The invention is illustrated by drawings.

Figure 1 shows a test tube with a coating coating 1). Test tube 2) is coated with a uniform layer of silicon oxide over the entire inner surface.

A vessel of plastic 1 (1) made of polypropylene or polyethylene coated polycarbonate inner walls 2. Application SiO ₂ SiO ₂ is carried out so that the container 2 remains optically transparent, and the layer thickness decreases with an increase in the internal volume of the vessel. This allows you to even out the heating rate of the liquid inside the vessel. The vessel can be made in the form of a test tube. Tubes without an inner coating have the same wall thickness throughout the volume. In view of the conical shape, the heating of the liquid in the test tubes without applying the inner coating is uneven.

To create a functional thin-film coating on the inside of polypropylene, polycarbonate and polyethylene tubes, from 0.2 to 50 ml; used for biochemical, molecular biological, genetic, cytological and other studies, thin film formation (synthesis) methods are used (such as thermal deposition, electron evaporation, gas deposition, electrolytic deposition, cathode spot deposition, magnetron evaporation, ion-ion plasma deposition, pulsed laser deposition, etc.). For example, when using the ion-plasma method:

- the tubes are located open side in the direction of flow of the sprayed material. The thin-film silicon oxide layer was synthesized by sputtering a silicon oxide target with a stream of high-energy Ar + ions (1000–1500 eV). Previously, the tubes are chemically cleaned with alcohol in an ultrasonic bath. The substrate holder allows you to get up to a thousand samples in one cycle (~ 1 hour).

The thickness of the silicon oxide synthesized on the surface of the tube (~ 2–400 nm) is sufficient for sorption of nucleic acids on the inner walls and to ensure a high degree of transparency of the obtained samples of plastic tubes.

Due to the optimal ion energy in this method, the obtained prototypes have high adhesion and a uniform thin-film coating.

With such combinations of parameters, the required transparency is achieved for real-time polymerase chain reaction (NDP) using fluorescent dyes, including intercalating ones.

Carrying out the described process of thin-film synthesis of SiO ₂ in ultra-high vacuum conditions results in ultrafine silicon oxide nanocoatings on the inner side of the tubes, with a volume of 0.2 to 50 ml, used for biochemical, molecular biological, genetic, cytological and other studies. Debugging the synthesis process leads to the selection of the most optimal energies of the condensing particles, which makes it possible to obtain SiO ₂ coatings with a high degree of adhesion to the inner surface of the tubes, while maintaining the transparency necessary for real-time PCR using fluorescent dyes, including intercalating ones. Coating can be done on the installation of ion-plasma spraying.

Thus, all the physical parameters of the tube are preserved (transparency, heat capacity, hardness) and new ones are acquired - the ability to absorb nucleic acids on the inner walls of the tube.

The method is as follows.

In a test tube made of polyethylene, polypropylene or polycarbonate with an inner surface coated with SiO ₂ , a sample of biological material or gel containing DNA fragments in an amount of 10-20000 mg, depending on the volume of the used tube, is placed. From 2 to 10 volumes of a solution of 1 containing a chaotropic reagent, a lysing reagent, salts of monovalent cations, a reagent for stabilizing the pH, a reagent for chelation, a reagent for inhibiting enzymatic activity are added. Perhaps the initial addition of a solution followed by the introduction of biological material. Thoroughly mix / pipet / shake the tube containing the components. Select solution 2 from the tube.

The implementation of the method is not limited to the above examples. Various combinations of these reagents and salts of monovalent elements are possible. For example, some reagents may be excluded if the reagent has several of the indicated properties. It is possible to exclude a reagent with the indicated properties if this property is not critical for the biological object from which nucleic acids are isolated or purified. The pH of the final mixture should be in the range of 7.8-8.4 (for the isolation and purification of DNA), 4.5-5.5 (for the isolation and purification of RNA). The concentration of salts of monovalent cations in the final mixture should be in the range of 250-500 mm. The introduction of proteolytic enzymes is possible. When proteolytic enzymes are added from solution 1, reagents that inhibit enzymatic activity are excluded. It is possible to introduce other enzymes whose activity is aimed at the breakdown of nucleic acids or proteins, depending on the tasks.

An example implementation of a method for the isolation and purification of nucleic acids from a liquid medium is that:

Method 1. 10-50 μl of blood is placed in a test tube with the lysis and / or chaotropic reagent contained in it (for example, the use of guanidine thiocyanate, sodium dodecyl sulfate, lauroyl sarcosyl) in an amount of 100-180 μl. The contents of the tubes are thoroughly mixed. Sodium chloride is added to a final concentration of 200-300 mM. Mix the contents. The contents are drained. 180 μl of ethanol are added to the tube. Mixed. The contents are drained. 180 μl of ethanol are added to the tube. Mixed. The contents are drained. The tube is dried. A mastermix mixture is added for PCR.

Method 2. A single cell with a small amount of medium is placed in a test tube. A buffer is added containing 10 mM Tris-chloride, 2 mM EDTA, 250 mM sodium chloride. The contents of the tube are heated to 70 ° C for 5 minutes. Mixed. The contents of the tube are drained. Mastermix for PCR is added.

If the isolated nucleic acids are intended for further enzymatic amplification by PCR, reverse transcription, restriction, ligation, modification, then the isolated nucleic acids can be dissolved directly in mixtures to carry out these enzymatic reactions. If it is necessary to dissolve the nucleic acids, double-distilled water, water treated with DEPC or a solution that prevents degradation of nucleic acids is added to the tube. Nucleic acids are stored at minus 20 degrees Celsius, both in dissolved form and in the state sorbed on the walls of the tubes.

Method Characteristics

1. The percentage (of the total content of DNA or RNA in the sample) of extraction from mammalian tissue, including human, - not less than 70%, bacterial cells - not less than 75%, from plant tissues - not less than 70%, from food products - not less than 60%, from degraded biological material - not less than 80%;

2. The reproducibility of quantitative and qualitative indicators - 90%;

3. The content of residual proteins is not more than 5%.

Thus, in a vessel that uses nanocoating obtained by thin-film synthesis, all physical parameters of the vessel (transparency, heat capacity, rigidity) are retained and new ones are acquired - the ability to sorb nucleic acids onto the inner walls of the tube, while providing a high process speed, fewer operations purity of the obtained nucleic acids.

Claims (15)

1. A plastic vessel for sorption of nucleic acids from a liquid medium, comprising a coating of silicon oxide on the inner surface of the vessel, characterized in that the coating is a nanocoating made by thin-film synthesis, including ion-plasma spraying, realized at ultrahigh vacuum by sputtering a silicon oxide target the flow of ions Ar +, while the thickness of this coating is made within 2 ÷ 400 nm. 2. The vessel according to claim 1, characterized in that the plastic is selected from the group comprising polyvinyl chloride, polyethylene, polypropylene, polystyrene, polycarbonates, acrylonitrile-butadiene-styrene, polyurethanes, polyamides, thermoplastic elastomers, polysulfones and polyetheretherketones, as well as mixtures thereof. 3. The vessel according to claim 1, characterized in that the energy of the Ar + ion stream is selected 1000 ÷ 1500 eV. 4. The vessel according to claim 1 or 2, characterized in that the vessel is made optically transparent. 5. The vessel according to claim 4, characterized in that the inner surface of the vessel before spraying is chemically cleaned with alcohol in an ultrasonic bath. 6. The vessel according to claim 1 or 2, characterized in that the thickness of the nanocoating does not exceed 25 nm. 7. A method for the isolation and purification of nucleic acids from a liquid medium, including their sorption on the inner walls of a plastic vessel, washing from impurities and elution in saline, characterized in that silicon oxide nanocoating made as a sorbent is used as a sorbent. by thin-film synthesis, including ion-plasma spraying, realized at ultrahigh vacuum by sputtering a silicon oxide target with an Ar + stream, the thickness of this coating being made in the range of 2–400 nm. 8. The method according to claim 7, characterized in that they use a vessel with a coating thickness of silicon oxide with sizes from 1 to 25 nm. 9. The method according to claim 7, characterized in that a chaotropic reagent, a pH buffer are additionally added to the vessel made of plastic with thin-film deposition of silicon oxide, the mixture is shaken, then drained or removed with an automatic pipette, alcohol is added to the vessel in a volume equal to the nominal volume the vessels are shaken and drained. 10. The method according to claim 7, characterized in that after adding the eluting solution, the vessel is heated to 95 ° C to isolate DNA or to 65 ° C to isolate RNA, shake vigorously and select the nucleic acid solution in another container. 11. A method for the isolation and purification of nucleic acids from a liquid medium, including their sorption on the inner walls of a plastic vessel, washing from impurities and elution in saline, characterized in that silicon oxide nanocoating made as a sorbent is used as a sorbent. by thin-film synthesis, including ion-plasma spraying, realized at ultrahigh vacuum by sputtering a silicon oxide target with an Ar + stream, while the thickness of this coating is made in the range 2–400 nm, p At the same time, a chaotropic reagent, a pH buffer are added to the vessel, the mixture is shaken, then drained or removed with an automatic pipette, then alcohol is added to the vessel from plastic in a volume equal to the nominal volume of the vessel, shaken and drained. 12. The method according to claim 11, characterized in that a vessel with a coating thickness of silicon oxide with sizes from 1 to 25 nm is used. 13. The method according to claim 11, characterized in that after adding the eluting solution, the vessel from the plastic is heated to 95 ° C to isolate DNA or to 65 ° C to isolate RNA, it is shaken vigorously and the nucleic acid solution is taken into another container. 14. A method for the isolation and purification of nucleic acids from a liquid medium, including their sorption on the inner walls of a plastic vessel, washing from impurities and elution in saline, heating the vessel, characterized in that oxide coating is used as an sorbent on the inner walls of the plastic vessel. silicon, performed by thin-film synthesis, including ion-plasma sputtering, realized at ultrahigh vacuum by sputtering a silicon oxide target with an Ar + stream, while the thickness of this coating is made in 2-400 nm, in addition, a chaotropic reagent, a pH buffer are added to the plastic vessel, after adding the eluting solution, the vessel is heated to 95 ° C to isolate DNA or to 65 ° C to isolate RNA, the nucleic acid solution is shaken vigorously and the solution is taken to another container. 15. The method according to 14, characterized in that a vessel with a thickness of a coating of silicon oxide with sizes from 1 to 25 nm is used.