RU2828813C9 - Method of purifying and recovering high-molecular nucleic acids, gel and device for implementation thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology and genetics. Described is a method of purifying and recovering high-molecular nucleic acids from a sample. Electrophoresis channel zone is filled with an agarose separating gel; applying a sample containing a target high molecular weight nucleic acid on an agarose separation gel; purification of high-molecular nucleic acid by electrophoresis; stopping electrophoresis after separation of high-molecular nucleic acid from contaminants; filling the gap between the gels with a low ionic strength electrophoresis buffer; resuming electrophoresis until the target nucleic acid migrates into the gap between the gels; collecting purified high-molecular nucleic acid from a gap between gels. Described also is a device for purifying and recovering high-molecular nucleic acids from a sample, comprising: at least one electrophoresis channel; zone of electrophoresis channel for filling with agarose separating gel containing buffer for electrophoresis with low ionic strength; zone of electrophoresis channel for installation of agarose blocking gel with content of salt in limits of 0.5 1 M of sodium chloride, gap between zone for filling with agarose separating gel containing buffer for electrophoresis with low ionic strength, and a zone for installing an agarose blocking gel with a salt content in range of 0.5 1 M sodium chloride; wherein the gap between the zone for filling with the agarose separating gel containing the buffer for electrophoresis with low ionic strength, and the zone for installation of the agarose blocking gel is made with possibility of filling with the buffer for electrophoresis with low ionic strength and is not less than 0.5 cm. in simplification of method for isolation and purification of high-molecular DNA or RNA, suitable for sequencing with long reading, high efficiency of extracting high-molecular DNA or RNA from various complex biological samples, for example, plant samples, soil samples, faeces, and so forth in improvement of quality of purification of high-molecular nucleic acids for sequencing with long reading, in acceleration of extraction and purification of high-molecular nucleic acids, in absence of need for additional purification of nucleic acids from salt, as well as providing the possibility of simultaneous processing of a large number of samples, which enables to scale and robotize the proposed technical solutions.

EFFECT: due to this it becomes possible, in particular, to obtain DNA or RNA with high degree of purity from 50 % to 95 % and high output from 50 % to 90 %.

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Description

The group of inventions relates to the field of biotechnology and genetics, in particular, to a method for purifying and isolating high-molecular nucleic acids DNA or RNA from biological samples for subsequent long-read sequencing, to a device and gel for purifying and isolating nucleic acids.

BACKGROUND OF THE INVENTION

Nowadays, purification and extraction of DNA or RNA from multicomponent biological samples represents the first step in a wide range of molecular biological protocols used in genetics, molecular medicine, forensics and biotechnology. The ultimate success of many of these protocols depends on the efficient separation of target nucleic acids from various impurities such as polypeptides and proteins (e.g. nucleases), oligonucleotides, polysaccharides, polyphenols, lipids, pigments, secondary metabolites, humic substances, various enzyme inhibitors, etc. One particular application, the success of which largely depends on the purity of DNA, is the determination of their nucleotide sequence (sequencing). In the era of Sanger sequencing and then in the era of massively parallel next-generation sequencing (NGS), the isolation of highly purified DNA was an important prerequisite for obtaining good results. Currently, the extraction of highly purified DNA remains a critical requirement for successful sequencing using third-generation sequencing (TGS) platforms that can read tens or even hundreds of thousands of base pairs. However, to take full advantage of such long reads, the DNA must not only be of high quality but also of the longest possible length. Thus, any long-read NGS or TGS method must combine high-molecular-weight DNA with high purity. Several approaches have been explored to address this issue.

LEVEL OF TECHNOLOGY

Numerous experimental protocols for DNA extraction and purification have been described (1-5). These protocols typically involve a cell lysis step followed by separation of DNA from contaminants. The most commonly used separation strategies can be divided into three categories: DNA precipitation, liquid-phase extraction, and solid-phase extraction.

The first separation strategy is based on the ability of polar solvents (e.g., ethanol) and salts (e.g., sodium acetate) to precipitate DNA from aqueous buffers (6). The actual DNA precipitation may be preceded by additional purification steps, such as protein denaturation and insolubilization (7). Although this strategy is suitable for the purification of high-molecular-weight DNA and is even used in commercial kits, the yield and purity of high-molecular-weight DNA may be suboptimal, especially for complex samples. In liquid-phase extraction, polar DNA remains in the aqueous phase, while non-polar contaminants, such as denatured proteins and lipids, are transferred to the organic phase (usually phenol-chloroform) or concentrated at the interface (8). To obtain DNA of acceptable purity, the organic extraction procedure must be repeated, which results in a decrease in yield. In solid-phase extraction, DNA is bound to a solid sorbent, and contaminants are removed by washing. Although many types of solid sorbents can be used for DNA purification, silica sorbents have become particularly popular in the last few decades. Commercial solid-phase extraction kits are widely available, and their advantages include reduced DNA extraction time and parallel processing of large numbers of samples. However, these kits also have their drawbacks: DNA extraction with them occurs in several steps and requires the use of commercial spin columns as well as specialized equipment such as centrifuges or vacuum filtration devices. Moreover, conventional solid-phase extraction protocols and commercial kits based on them are generally not suitable for the extraction of high-molecular-weight and highly purified DNA. When using these protocols, a large part of the extracted DNA bypasses the column and is not adsorbed, which ultimately leads to low yields. Thus, a number of methods are available for the extraction and purification of high-molecular-weight DNA, but they all have individual advantages and limitations, such as low yields of final DNA, limitations in molecular size (due to mechanical degradation of high-molecular-weight DNA), low purification efficiency (since polysaccharides and other high-molecular-weight compounds remain bound to DNA), cost (for purification on magnetic beads), and the possible use of toxic chemicals (phenol, chloroform, thiocyanates). Most likely, at present there is no universal method for the extraction of high-molecular-weight DNA that would be independent of the nature and chemical composition of the sample. The lack of such a method is especially problematic if the starting material is a complex mixture of chemically different molecules containing only a small amount of the target DNA.

One approach to extract high-quality DNA from complex mixtures is gel electrophoresis followed by electroelution from excised gel fragments containing the target DNA. The most common variant of this method is electroelution in dialysis bags (9,10). Other variants, such as glycerol gradient electroelution (11), have also been described in the literature. However, the considerable labor intensity of these traditional methods limits their applicability in routine laboratory practice. Electrophoresis followed by continuous electroelution (12, 13) is an interesting alternative solution that can obtain highly purified DNA from gels. Such DNA will be compatible with most molecular biology applications. One example of such an approach is the Sage ELF system (Sage Science, Inc., USA), in which DNA is loaded into an agarose gel cassette and, after electrophoresis, the separated DNA fragments are electroeluted from the gel using side electrodes into twelve separate sample collection wells. The resulting DNA fractions can be used for nucleotide sequencing and other applications without further purification. Although Sage ELF is a good tool for DNA fractionation and purification, its disadvantages include expensive equipment and consumables, an upper limit of 40 kb on the mass of isolated fragments, and the inability to simultaneously process large numbers of samples.

The prior art includes patent for invention RU 2650865 dated 17.04.2018 (45), which describes a reagent kit for isolating DNA from biological objects. The kit includes a lysis buffer solution, wash buffer solutions No. 1 and No. 2, and an eluting buffer solution. The lysis buffer solution contains: a strong chaotropic agent - guanidine thiocyanate, non-ionic detergent Triton X-100, a pH buffer based on tris-hydrochloride, and a sorbent in the form of a suspension of magnetic microspheres in a saline solution. Wash buffer No. 1 contains guanidine thiocyanate, tris-hydrochloride and ethyl alcohol. Wash buffer No. 2 contains tris-hydrochloride, sodium chloride and ethyl alcohol. The eluting solution is deionized water. The invention provides an increase in the yield of DNA.

Known is the patent for utility model RU 84381 U1 from 10.07.2009 (46), which describes a device for the automated extraction of nucleic acids from biological samples, consisting of (a) a cartridge containing reservoirs with reagents for lysis of cells, microorganisms and viral particles, purification and elution of nucleic acids, channels, microcolumns and valves, and (b) a control unit-controller, implementing pressure supply, heating, mixing of reagents in individual reservoirs of the cartridge and movement of reaction mixtures and solutions in the reservoirs of the cartridge. The cartridge comprises a receiving chamber for introducing a biological sample, reservoirs with dry reaction mixtures of lysis buffers and a washing buffer, a microcolumn with a solid-phase sorbent for binding nucleic acids, reservoirs for ethanol, a water-ethanol and water mixture for dissolving dry components of the washing buffer, washing the microcolumn and eluting nucleic acids bound to the solid-phase sorbent in the microcolumn, an output port compatible with a standard microtube, a reservoir for collecting reaction waste, and the control unit-controller comprises a solenoid block for controlling the cartridge valves, a heater block, an electromagnetic stirrer block, a compressor and an electronic control unit.

The disadvantages of the above-mentioned patented technical solutions are the long duration and multi-stage nature of the DNA purification procedure, low DNA yield and low degree of purification for “complex” samples.

The source of information is known - the Zarzosa-Alvarezetal protocol. (19), where the excised fragment of agarose gel containing the target DNA is placed in an electroelution device (electroeluther), and its V-shaped channel is filled with a buffer with an increased salt content (1 M sodium chloride). During electroelution, the DNA fragment migrates from the agarose gel into a salt trap. At the final stage of the protocol, the buffer with an increased salt content containing the electroeluted DNA is removed with a pipette and precipitated with ethanol. The specified source of information was chosen as the closest analogue (prototype). The disadvantage of the prototype is that this method is quite labor-intensive, namely, it requires excising DNA bands from the agarose gel under UV-A radiation, transferring the resulting agarose fragment to the electroeluther and separately performing the electroelution process. Thus, this approach only allows for a DNA purification step and requires special equipment (electroeluther). Additionally, a disadvantage of this method is that the target DNA is dissolved in a high-salt buffer and is unsuitable for further use without additional purification. Precipitation of DNA from a salt solution is mandatory, which in turn can lead to a decrease in DNA yield with a highly diluted solution.

As a result of the analysis of the state of the art, it can be concluded that the proposed group of inventions can be recognized as meeting the patentability criteria of “novelty” and “inventive step” before the date of the requested priority.

DISCLOSURE OF THE ESSENCE OF THE INVENTION

In order to solve the technical problem, as well as to eliminate the shortcomings of the existing analogues and prototype, the applicant has proposed a method and device for purifying and isolating high-molecular DNA or RNA from biological samples, as well as a gel for purifying and isolating high-molecular DNA or RNA and a method for manufacturing the device. The group of inventions is united by a single inventive concept.

Long-read sequencing technologies require DNA not only of high purity and integrity, but also of high molecular weight, which can be difficult to obtain from complex biological samples.

In this regard, the authors have developed a method for purifying and isolating high-molecular DNA that includes electrophoresis followed by electroelution and is based on the fact that the electrophoretic mobility of nucleic acids slows down in gels and solutions with a high ionic strength. A sample of high-molecular DNA is first purified by electrophoresis in a separating channel filled with a gel (e.g., agarose). After sufficient purification, electrophoresis is stopped and a blocking gel containing an increased salt concentration is placed in front of the DNA band (downstream of its direction of movement). The purified high-molecular nucleic acid can be collected from a reservoir (gap) for collecting samples located between the main gel and the gel containing an increased salt concentration. After installing the blocking salt gel, the current is applied repeatedly until the DNA is electroeluted from the separating gel into the collection reservoir (gap between the gels), where it gradually slows down and accumulates. The purified DNA can be easily collected from the reservoir using a pipette and used without additional desalting in long-read sequencing reactions. The advantages of this method are that it is simple and inexpensive, and allows for the production of high-molecular DNA or RNA of high purity and high yield even from complex biological samples. Other advantages are that the proposed method is compatible with long-read sequencing and has the potential for automation in vertical or horizontal format.

The purpose of the proposed technical solution is to create a method, device and components for the implementation of high-quality purification of high-molecular nucleic acids from multicomponent biological samples for the purpose of their subsequent sequencing by third-generation methods. There is a need to develop new methods for isolating and purifying DNA from biological samples, and new, more effective devices and materials for implementing such methods.

The technical result consists in simplifying the method for isolating and purifying high-molecular DNA or RNA suitable for long-read sequencing, increasing the efficiency of isolating high-molecular DNA or RNA from various complex biological samples (e.g. plant samples, soil samples, feces, etc.), improving the quality of purifying high-molecular nucleic acids for long-read sequencing, accelerating the isolation and purification of high-molecular nucleic acids due to the absence of the stage of transferring the material from the electrophoresis device to the electroeluther, in the absence of the need for additional purification of nucleic acids from salt, as well as ensuring the possibility of simultaneously processing a large number of samples in the proposed device using the claimed method, which allows scaling and robotizing the proposed technical solutions. Due to this, it becomes possible, in particular, to obtain DNA (or RNA) with a high degree of purity (from 50% to 95%) and a high yield (from 50% to 90%).

OBJECT OF INVENTION

The present group of inventions describes a simple and effective method for purifying and isolating high-molecular DNA (or RNA), a device and gel for purifying and isolating high-molecular DNA (or RNA), and a method for manufacturing the device.

The specified technical result is achieved by a set of essential features presented in the invention formula.

In one non-limiting embodiment of the invention, a method is provided for purifying and isolating high molecular weight nucleic acids from a sample, comprising:

- filling the electrophoresis channel zone with an agarose separating gel containing an electrophoresis buffer with low ionic strength;

- application of a sample containing the target high-molecular nucleic acid to an agarose separating gel containing a low ionic strength electrophoresis buffer;

- purification of high molecular weight nucleic acid by electrophoresis;

- stopping electrophoresis after separation of high-molecular nucleic acid from contaminants until the purified nucleic acid exits the agarose separating gel containing electrophoresis buffer with low ionic strength;

- installation of an agarose blocking gel with a salt content in the range of 0.5 - 1 M sodium chloride in the electrophoresis channel in the direction of movement of nucleic acids, after an agarose separating gel containing a buffer for electrophoresis with a low ionic strength, in which the purified nucleic acid is located, with a gap between the gels of at least 0.5 cm;

- filling the gap between the gels with electrophoresis buffer of low ionic strength;

- resumption of electrophoresis until the target nucleic acid migrates into the gap between the gels;

- selection of purified high-molecular nucleic acid from the gap between the gels.

In another example of implementing the invention, the selection of purified nucleic acid from the gap between the gels is carried out using an automatic pipette or a laboratory robot.

In another example of implementing the invention, the purified nucleic acid is collected from the gap between the gels into a microtube for further use.

In another example of implementing the invention, before purifying a high-molecular nucleic acid using electrophoresis, the duration of electrophoresis is determined.

In another embodiment of the invention, the gap between the main and agarose blocking gels is filled with a low ionic strength electrophoresis buffer. Low ionic strength is understood to mean an ionic strength lower than that of the agarose blocking gel.

In another embodiment of the invention, the low ionic strength electrophoresis buffer is 20 mM Tris and 20 mM HEPES.

In another example of implementing the invention, the sample additionally contains a mixture of biological molecules including non-target nucleic acids or short oligonucleotides, or proteins, or peptides, or polysaccharides, or lipids, or secondary metabolites, or humic substances, or pigments, or polyphenols.

The technical result is also achieved due to the fact that the device for purifying and isolating high-molecular nucleic acids from a sample includes:

- at least one electrophoresis channel;

- a zone of the electrophoresis channel for filling with an agarose separating gel containing an electrophoresis buffer with a low ionic strength;

- an electrophoresis channel zone for installing an agarose blocking gel with a salt content in the range of 0.5 - 1 M sodium chloride,

- a gap between the zone for filling with an agarose separating gel containing a low ionic strength electrophoresis buffer and the zone for installing an agarose blocking gel with a salt content in the range of 0.5 - 1 M sodium chloride;

- wherein the gap between the zone for filling with an agarose separating gel containing a buffer for electrophoresis with a low ionic strength and the zone for installing an agarose blocking gel is designed with the possibility of filling with a buffer for electrophoresis with a low ionic strength and is at least 0.5 cm. In another example of implementing the invention, the electrophoresis channel is made horizontal or vertical.

In another example of the invention, the electrophoresis channel is made horizontal or vertical.

In another example of the invention, the electrophoresis channel is made open or closed.

In another embodiment of the invention, the low ionic strength electrophoresis buffer is 20 mM Tris and 20 mM HEPES.

In another example of the invention, the device is made of plastic that is transparent to visible and UV radiation, for example, 315-400 nm.

In another example of implementing the invention, the device additionally includes electrodes for performing electrophoresis in the electrophoresis channel.

In another example of implementing the invention, the device additionally includes a sensor for collecting information about the isolated high-molecular nucleic acids, connected to the information processing unit and the display unit.

BRIEF DESCRIPTION OF FIGURES, TABLES, AND OTHER GRAPHIC MATERIALS

The invention is illustrated by the following graphic materials.

Figure 1 shows the principle of trapping electroeluted DNA in the gap between low and high ionic strength gels.

Figure 2 shows a schematic diagram of the method (workflow overview) for high molecular weight DNA gel purification.

Figure 3 shows a photograph of the GeneRuler DNA LadderMix (ThermoFisherScientific, SM0332) for determining the size and approximate quantification of double-stranded DNA on agarose gels and compares the results of DNA purification by a method known from the prior art (using the method described in the prototype) and DNA purified using the claimed method.

Figure 4 shows a model of a device for purifying and isolating high-molecular nucleic acids from a sample.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and general terminology.

Reference will now be made to specific embodiments of the invention, examples of which are illustrated by the proposed method and device. It is understood that the invention covers all alternatives, modifications and equivalents that may be included within the scope of the present invention, as defined in the claims.

A person skilled in the art knows many methods and materials similar or equivalent to those described in this document, which can be used in the practical implementation of the present group of inventions.

The present invention is not limited to the methods and materials described herein. In the event that one or more of the incorporated literature references, patents, and similar materials differ from or contradict this application, including, but not limited to, defined terms, use of terms, described techniques, etc., this application shall prevail.

It should further be appreciated that certain features of the invention, which for clarity are described in the context of separate embodiments of the invention, may also be presented together in a single embodiment. Conversely, various features of the invention, which for brevity are described in the context of a single embodiment of the invention, may be presented separately or in any suitable subcombination.

Where a range of values is presented, it is to be understood that each intermediate value, up to tenths of a unit of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limits of this range and any other stated or intermediate values in this stated interval are encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, which are also within the scope of the invention, taking into account any specifically excluded limit in the stated range. When a range is stated to include one or both limits, ranges excluding either of both included limits are also within the scope of the invention.

All publications, patents and patent applications are incorporated herein by reference. Although the invention has been described in the foregoing description with respect to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that some details described herein may be substantially modified without departing from the spirit of the invention.

The use of singular terms in the context of the description of the invention shall be construed as covering both the singular and the plural unless otherwise indicated herein or clearly contradicted by context.

The terms “consisting of,” “having,” “including,” and “containing” are to be construed as open-ended terms, i.e., meaning “including, but not limited to,” unless otherwise specified.

The use of any and all examples or illustrative language (e.g., "such as") provided herein is intended merely to better describe the invention and does not limit the scope of the invention unless otherwise stated.

No statement in the description should be construed as indicating any non-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these embodiments may become apparent to those skilled in the art upon reading the preceding description. The inventors expect skilled artisans to employ such variations as the circumstances dictate, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the features set forth in the appended claims as permitted by applicable law. Moreover, any combination of the above-described features in all their possible variations is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

The following terms are used to describe the present invention.

All technical and scientific terms used in this document have the same meaning as commonly understood by those skilled in the art to which the invention pertains, unless otherwise indicated. All patents and publications referenced in this document are incorporated by reference in their entirety.

The abbreviations used in this description, including those given in the illustrative diagrams and the following examples, are well known to those of ordinary skill in the art. Some of the abbreviations used are as follows:

DNA - deoxyribonucleic acid.

RNA - ribonucleic acid.

Non-target nucleic acid is impurity nucleic acid that is not the object of purification and isolation.

Electrophoresis channel – a channel in a device with the ability to perform electrophoresis, which is subjected to electrophoresis.

NGS - next generation sequencing.

HEPES - (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is a zwitterionic organic buffering agent; one of twenty in Good's list of buffer solutions.

THE - electrophoresis buffer (Tris-HEPES-EDTA), also for dissolving and storing DNA, 1x buffer contains 20 mM Tris and 20 mM HEPES and 0.1 mM EDTA.

TE - buffer for DNA dissolution and storage, 1x buffer contains 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA.

TBE – electrophoresis buffer (Tris-borate-EDTA), 1x buffer contains 45 mM Tris, 45 mM boric acid and 0.5 mM EDTA, pH 8.3.

TAE - electrophoresis buffer (Tris-acetate-EDTA), 1x buffer contains 40 mM Tris, 20 mM acetate and 0.5 mM EDTA, pH 8.0. 5 l packaging is supplied in a cubic container.

EDTA - ethylenediaminetetraacetic acid (from English EDTA).

SDS - sodium dodecyl sulfate, an anionic surfactant.

CTAB - cetyltrimethylammonium bromide, a synthetic surfactant, has antiseptic and antistatic effects.

TGS is a third-generation sequencing platform.

SYBR GreenI is an asymmetric cyanine dye used in molecular biology to stain nucleic acids.

EtBr - ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridium bromide), a fluorescent dye used as an intercalating agent in molecular biology for detecting nucleic acids during DNA electrophoresis in agarose gel.

XyleneCyanol FF is a xylene cyanol used as a tracking dye in agarose or polyacrylamide gel electrophoresis. It has a slight negative charge and migrates in the same direction as the DNA, allowing the user to follow the molecules as they move through the gel. The rate of migration depends on the composition of the gel.

PFGE - pulsed field gel electrophoresis.

EXPERIMENTAL PROCEDURES

The claimed method for isolating and purifying high-molecular nucleic acids from a sample involves filling the electrophoresis channel zone with a basic gel, then applying the sample to a slot (well for applying the sample).

Next, the high-molecular nucleic acid is purified by electrophoresis. Then, the electrophoresis is stopped after the high-molecular nucleic acid has been separated from contaminants and before the purified nucleic acid has exited the main gel. Then, an agarose blocking gel with an increased salt content is installed in the electrophoresis channel after the main gel containing the purified nucleic acid, creating a gap between the gels of at least 0.5 cm. At the next stage, the gap between the gels is filled with electrophoresis buffer and electrophoresis is resumed until the target nucleic acid migrates into the gap between the gels. At the last stage of the method, the purified high-molecular nucleic acid is collected from the gap between the gels.

The claimed method includes a gel electrophoresis method followed by electroelution. It is based on the fact that the electrophoretic mobility of DNA decreases with increasing salt concentration (ionic strength) in the electrophoresis medium. After sufficient separation of DNA from various contaminants, a gel block with an increased salt content is placed in the path of further DNA (or RNA) electrophoresis, and a reservoir for collecting the purified sample is located between the separating gel and the gel with an increased salt content. Electroelution from the main (separating) gel can continue until the target DNA migrates into the collection reservoir (the gap between the gels), where it gradually slows down and accumulates. Purified DNA can be easily removed from the reservoir using a pipette and used either immediately or after desalting, depending on further application. The proposed DNA purification method is especially useful when other methods are ineffective or impractical, for example, for processing multicomponent samples containing complex mixtures of biomolecules. In addition, the claimed method is cost-effective and does not require complex reagents or tools.

In ionic solutions, the negatively charged nucleic acid is surrounded by positively charged cations (counterions) and negatively charged anions (co-ions), which form the so-called ionic atmosphere. The ionic atmosphere has a higher concentration of attracted cations (counterion accumulation) and a lower concentration of repelled anions (co-ion depletion) and is strongly dependent on the ionic strength of the solution.

It is known that an increase in ionic strength causes electrostatic shielding of the negative charge of DNA by counterions (Fig. 1), thereby reducing its mobility in solution during capillary electrophoresis.

Figure 1 shows the principle of trapping electroeluted DNA in the gap between low and high ionic strength gels (Fig. 1 example 3). Schematic representation of the ionic atmosphere surrounding DNA in gels containing different salt concentrations. Red and blue circles represent Na ⁺ cations and Cl ^- anions. The higher accumulation of cations around DNA and RNA in the high salt gel (Fig. 1 example 2) compared to the normal gel (Fig. 1 example 1) causes stronger electrostatic shielding of the negative charge of DNA, thereby reducing its electrophoretic mobility. This phenomenon can be used to trap DNA in the buffer-filled gap in front of the high salt gel, where DNA mobility slows down and accumulation occurs (Fig. 1 example 3).

Based on the principle underlying the above methodology, that the electrophoretic mobility of nucleic acids is dependent on the salt concentration and can be greatly reduced in the presence of a high salt concentration when a salt block (blocking gel) containing an increased salt concentration is positioned in the direction of migration of the nucleic acids. To demonstrate this phenomenon, a section of gel ahead of the migrating DNA was excised and replaced with a gel containing an increased salt concentration. When approaching the high salt gel (agarose blocking gel), the DNA showed a marked decrease in electrophoretic mobility, indicating electrostatic shielding by excess counterions (Fig. 1). Based on this proof of concept, a method for purifying high molecular weight DNA (or RNA) was developed that combines gel electrophoresis and electroelution and involves trapping the DNA (RNA) with a gel block (by adding an agarose blocking gel) with an increased salt content.

The proposed method and device use high-molecular DNA purification by electrophoresis, for example, in horizontal channels. The gel molding tray can be easily manufactured independently using 3D printing or on a milling machine for processing a plastic blank transparent to visible and UV radiation. The width and height of each channel can be approximately 1 cm, which is sufficient for the effective separation and purification of standard DNA samples and for placing a single-well comb for electrophoresis.

Fig. 2 schematically shows the process of purification of a high molecular weight DNA gel proposed in the invention.

Two sections were cut downstream of the sample deposition site, creating a reservoir for collecting the cleared sample (gap) and a molding cavity for placing the agarose blocking gel.

In the next step, the resulting molding cavity can be filled with a molten solution of agarose blocking gel containing an increased concentration of salt. In one embodiment of the method, after the agarose blocking gel with an increased salt content hardened, it was carefully removed from the molding tray and set aside for further use during electrophoresis.

The tray (reservoir for placing the agarose blocking gel, see Fig. 4), which now contained only the main gel, was transferred to the electrophoresis reservoir.

A sample containing the target high molecular weight DNA was loaded into a well on the base gel and subjected to electrophoresis.

Electrophoresis was continued until the high molecular weight DNA reached the end of the base gel, the current was temporarily turned off and the blocking gel was removed from the electrophoresis reservoir.

The sample collection reservoir (gap) was filled with fresh low ionic strength electrophoresis buffer. The agarose blocking gel was installed and the current was turned on again to electroelute the purified high molecular weight DNA from the separating gel into the reservoir.

Further migration of DNA through the buffer-filled gap was gradually slowed by neutralization of the negative charge of DNA by excess cations moving towards it from the agarose blocking gel. This resulted in the accumulation of purified high-molecular-weight DNA in the gap, from which it could be easily extracted with a pipette.

Gel electrophoresis is the method of choice for purifying long oligonucleotides because it offers one of the highest levels of purity among available purification methods. The same is true for the purification of full-length DNA.

The result of using the claimed method is shown in Fig. 3, where effectively purified and concentrated DNA is observed.

The method proposed in this invention does not involve grinding the gel; instead, it is based on a continuous process of electrophoresis and electroelution that does not disrupt the structure of the gel, thereby eliminating contamination of the sample with polysaccharides from agarose.

In the proposed method, electroeluted DNA is collected in a sample collection reservoir (the gap between the gels) filled with an electrophoresis buffer. Preferably, the buffer should be compatible with subsequent enzymatic reactions and long-term storage of samples. Tris-borate-EDTA (TBE) electrophoresis buffer is one of the most common working buffers used in electrophoresis of short nucleic acid fragments. However, borate ions form complexes with DNA (22, 23) and proteins (24-26), and inhibit the activity of many enzymes (27). This makes TBE unsuitable for this protocol. The most popular alternative to TBE is Tris-acetate-EDTA (TAE) buffer. TAE buffer, based on acetic acid and Tris, can be used. However, the high acetic acid content of TAE buffer can potentially inhibit some enzymatic reactions such as PCR (28) and the buffer rapidly loses buffering capacity and heats up with prolonged use. The presence of high concentrations of EDTA in buffers can also inhibit PCR (28) and a number of DNA modifying enzymes such as alkaline phosphatase (29) and nuclease S1 (30).

Determining the localization of the target high-molecular-weight DNA band of interest is important in determining when to use a high-salt blocking gel to stop the mobility of DNA and RNA. The easiest way to estimate (without using UV-A light) when the high-molecular-weight DNA is approaching the end of the separating gel is to use a slow-migrating lead dye such as XyleneCyanol FF. Since XyleneCyanol migrates in a 0.8% agarose gel at approximately the same rate as a dsDNA fragment of at least 10 kb, the high-salt blocking gel can be used after the XyleneCyanol dye begins to migrate out of the main gel.

Alternatively, it is possible to assess the migration of high molecular weight DNA based on the position of fragments pre-stained (e.g. stained with intercalating dyes (SYBR Green I, EvaGreen, SYBR Safe, GelRed or GelGreen)) using e.g. near UV-A radiation (LED black light source in the range of 365-395 nm), which will not destroy nucleic acids. The most common way to detect DNA in a gel is to use a fluorescent intercalating dye, a small molecule that reversibly intercalates between adjacent base pairs of the double helix. Ethidium bromide (EtBr) remains the most widely used fluorescent intercalating dye in DNA electrophoresis despite the fact that it is a potential carcinogen and the preferred use of UV-B (210 nm and 285 nm) radiation for visualization results in DNA damage. Using e.g. near UV-A radiation (LED black light source) allowed when staining DNA with EtBr dye. Alternatives to EtBr dye include, among others, SYBR Green I, EvaGreen, SYBR Safe, GelRed and GelGreen, all of which have better safety profiles than EtBr and can be visualized using, for example, near-UVA radiation (black light source, in the range of 365-395 nm) or blue light. However, due to their DNA-binding nature, all intercalating dyes alter the structure and mechanical properties of DNA (31-33) and will potentially interfere with subsequent enzymatic reactions (34-36). Therefore, if the subsequent application is sensitive to the presence of an intercalating dye, an additional purification step is recommended, using isopropanol DNA precipitation (see paragraph below). However, the simplest way to avoid the problem described above is to first determine the time at which the fluorescently stained high-molecular-weight DNA reaches the end of the separating gel, and then routinely use the obtained parameters (electrophoresis time and voltage used) to isolate similar, but unstained, DNA. This approach can be especially valuable when parallel isolation of high-molecular-weight DNAs of the same size is performed in large-scale experiments.

A balance must be struck when selecting the salt concentration in the blocking gel. On the one hand, the salt concentration must be sufficient to slow down the migration of DNA in the gap between the gels. On the other hand, too high a salt concentration will contaminate the DNA sample and may cause DNA to be arrested in the separating gel before it reaches the sample collection reservoir. After a series of tests, the optimum concentration (i.e., the additional increase in the technical result) of salt was found to be approximately 0.5 - 1 M sodium chloride. This value is consistent with previously published data showing that the electrophoretic mobility of double-stranded DNA in free solution decreased with increasing ionic strength until it began to level off at approximately 0.6 M sodium acetate (16). With 0.5 M sodium chloride in the blocking gel, the sodium ion concentration of the isolated DNA sample was between 30 and 40 mM as determined by inductively coupled plasma optical emission spectroscopy (ICP-OES). Most enzymatic reactions, even the most salt-sensitive ones (e.g., ligation with T4 DNA ligase), are compatible with this salt concentration, especially considering the sample dilution in the reaction mixture. Thus, the obtained DNA is suitable without additional desalting for a variety of subsequent enzymatic reactions, including adapter ligation for long-read sequencing. However, for some specific applications, such as sample preparation for sequencing using third-generation platforms, for DNA analysis by laser desorption/ionization (MALDI) mass spectrometry (37), an additional desalting step and sample concentration may be required. Precipitation of nucleic acids by mixing an equal volume of a salt mixture with isopropanol is the first choice method for desalting and concentrating DNA. This method requires a refrigerated benchtop centrifuge and a certain time for incubating the sample at -20° in a freezer, but may result in incomplete precipitation of DNA from dilute samples. Another approach for simultaneous desalting and concentrating of samples is to use commercial centrifugal membrane filter units (e.g., Amicon Ultra-2 CentrifugalFilterUnit from Millipore). Additionally, alternative desalting methods exist such as gel filtration on spin columns followed (if necessary) by butanol extraction (38) to concentrate the sample. The spin column can be either commercially available or packed with gel filtration sorbent (39) in-house. Thus, there are several methods for efficient desalting and concentrating of high molecular weight DNA. These methods, in addition to performing their primary functions, also separate any residual leading and/or intercalating dyes from the target high molecular weight DNA.

The main advantage of the proposed method is that it provides a simple and efficient solution for the extraction and purification of high-molecular-weight DNA suitable for long-read sequencing. Current long-read/TGS technologies (40) were developed to overcome the main limitation of second-generation sequencing, namely short reads of less than 600 nucleotides in length. Longer reads (ranging from 10 kb to more than 400 kb) generated by TGS significantly improve the quality and completeness of genome assembly and are particularly useful for characterizing highly repetitive genomic regions. The current reaction mixture used in TGS is sensitive to the presence of contaminants in DNA samples. Therefore, it is important to obtain DNA with both high molecular weight and high purity to take advantage of the opportunities offered by TGS.

Although the upper limit of resolution of the 0.8% agarose gels used in the present study is approximately 50 kb, larger DNA molecules can also be recovered from such gels if the DNA enters and migrates within the gel in a process called reptation (41). Thus, the proposed method can be successfully used to separate DNA larger than 50 kb from smaller contaminants. However, if there is a need to separate individual DNA molecules larger than 50 kb from each other, the upper limit of resolution of gel electrophoresis must be extended. This can theoretically be achieved using low-percentage liquid agarose gels (0.1–0.3%), which are capable of separating DNA molecules up to 750 kb in length. However, such gels are fragile in horizontal mode, require special handling, and separation is time consuming (42,43). The use of short vertical and closed channels with a liquid gel allows good and fast elution of whole high-molecular DNA of any size (viruses, chromosomes). Another way to extend the upper limit of resolution of agarose gels is to use pulsed-field gel electrophoresis (PFGE), which is based on the application of an electric field to the gel from different directions. Unlike conventional electrophoresis in a steady field, PFGE is capable of separating DNA up to 5 million base pairs in length (44). The claimed method can be used to isolate high-molecular DNA separated by PFGE. From the point of view of electrophoresis theory, there are no obstacles to the use of a DNA trap based on a blocking gel with an increased salt content in combination with PFGE. Thus, the claimed method has the potential to isolate high-molecular DNA up to several million bases in size with a purity suitable for TGS and other demanding medical and technological applications.

Another advantage of the proposed method is the ability of gel electrophoresis to separate the DNA of interest from chemically diverse biomolecules in a single step. These molecules may include, but are not limited to, the following: unwanted long-chain DNA or RNA, short oligonucleotides of DNA and RNA, polysaccharides, polyphenols, humic substances, proteins, peptides, lipids, pigments, and secondary metabolites. Conventional DNA purification methods often require multiple steps when working with complex samples. In contrast, gel electrophoresis allows the separation of target DNA from various chemically related and unrelated molecules in a single step based on their differences in charge and size. In addition, gel electrophoresis typically does not require complex sample pretreatment to achieve good separation. In our experiments, a simple and inexpensive protocol based on sodium dodecyl sulfate and proteinase K, which preserves the primary size of high-molecular-weight DNA, was sufficient for sample processing. Another advantage of DNA purification by gel electrophoresis is that it requires less starting DNA than other purification methods. This is especially valuable when the purified sample contains only a small amount of target DNA. In addition, loading several micrograms of DNA for gel separation causes band blurring, a problem that becomes more severe as the DNA size increases. For best results, it is recommended to use less starting material than is typically required for other methods. For example, as little as 10 μg of a tissue sample with normal DNA content may be sufficient to obtain up to 1 μg of purified high-molecular-weight DNA. Accordingly, the proposed method is effective for one-step processing of complex samples containing chemically diverse impurities and only a small amount of target DNA.

An important feature of the proposed method is its scalability and automation potential. It is possible to scale the system to accommodate 8, 12, 24, 48 or even more separation channels in a vertical or horizontal version, and to optimize the channel shapes for specific high-performance tasks.

After initial optimization using 3D printing, a suitable prototype device can be CNC-fabricated from UV-A transparent plastic. Figure 4 shows a model of an electrophoresis/electroelution device for large-scale purification of high-molecular-weight DNA, having a channel with two extensions, the first of which contains a pre-cast base (separation) agarose gel. A sample containing the target DNA is loaded into the sample loading well and electrophoresis is carried out until the high-molecular-weight DNA reaches the end of the separating gel and all unwanted molecules have eluted from it. Electrophoresis is stopped, the collection reservoir for the purified sample is filled with fresh buffer, and a tray with base and blocking gel is inserted into the second extension of the electrophoresis channel. Current is applied repeatedly until the high-molecular-weight DNA migrates from the base gel into the buffer-filled collection reservoir, where it is decelerated and accumulated. The device can be scaled up to accommodate multiple separation channels. Much of the workflow can be automated, including loading samples, adding fresh buffer, introducing a high salt gel, and drawing DNA from the purified sample collection reservoir.

Thus, the proposed device and method for DNA extraction and purification are effective tools for the extraction of high-molecular DNA for long-read sequencing from eukaryotic and prokaryotic cells, mitochondria, chloroplasts, large viruses, etc. The device or method can be used when other methods are ineffective, for example, for single-step purification of high-molecular DNA from complex biological samples containing a large number of chemically diverse impurities and only a small amount of target DNA. In addition, the method is cost-effective and has the potential for scalability and a high degree of automation.

Sodium measurements were performed by inductively coupled plasma optical emission spectroscopy (ICP-OES) using an Agilent 720 instrument (Agilent Technologies, Inc., Santa Clara, CA, USA). The following sodium atomic lines were selected: 568.821 nm, 588.995 nm, and 589.592 nm. The average sodium content was calculated from two replicates. All samples were diluted 20-fold with ultrapure water before analysis. Precision was assessed using spiked sample standards according to ISO 5725-4 (2020) recommendations.

The 50x THE electrophoresis buffer used is 1 M Tris, 1 M HEPES (121.14 g Tris base, 238.3 g HEPES (free acid); dissolved in ultrapure water and made up to 1 liter of final volume). The pH does not need to be adjusted and should be between 8.0 and 8.1.

The sequence of implementation of the claimed method can be presented as follows.

1. Prepare a 2-liter bottle of 1x THE electrophoresis buffer by dissolving 40 ml of 50x THE buffer in a total volume of 2 liters of distilled water.

2. Prepare a 5 M NaCl solution, dissolve 292 g NaCl in ultrapure water and bring the final volume to 1 liter.

3. To prepare a 0.8% base separating gel, add 0.8 g of ultrapure agarose to 100 ml of 1x THE buffer in a 250 ml screw-cap glass bottle. The volume of the solution, referred to hereafter as agarose solution 1 (AS1), is more than enough to fill five gel channels.

4. Heat the flask with AS1 in a microwave oven at maximum power (e.g. 800 W), shaking gently from time to time, until all the agarose is completely dissolved, forming a clear solution. Be careful not to let the solution boil away. Store the melted agarose at 45-55°C for later use.

5. To prepare a 1% high salt blocking gel, add 1 g of ultrapure agarose to 90 ml of 1x THE buffer in a 250 ml screw cap glass bottle. The solution is referred to as agarose solution 2 (AS2).

6. Heat the flask with AS2 in a microwave oven at maximum power (e.g. 800 W), shaking gently from time to time, until all the agarose is completely dissolved, forming a clear solution. Make sure that the solution does not boil away. After the agarose is completely dissolved, add 10 ml of 5 M NaCl solution, mix well. Store the melted agarose at 45-55 ° C for further use.

7. Insert a single-well electrophoresis comb into the top of the gel-forming channel. The channel is sealed on both sides with adhesive tape, which is removed before starting electrophoresis. Pour the AS1 solution into the channel, filling the entire cavity of the channel, and ensure that no air bubbles get under the teeth of the comb. Allow the gel to harden at room temperature, forming a horizontal separation channel of agarose 1 cm wide and deep. The gel will become slightly opaque after complete hardening.

9. Using a clean scalpel, excise a 1 cm section of gel ~4 mm beyond the comb (this will be the gel that will be used for electrophoresis and DNA purification), creating a sample collection reservoir. Excise another 1 cm section of gel ~3 mm beyond the DNA sample collection reservoir, creating a mold for the high salt blocking gel. Pour the AS2 solution into the mold cavity and allow the blocking gel to harden at room temperature.

10. Carefully extract the high-salt blocking gel with a spatula, surrounded by short fragments of low-salt electrophoresis gel, for further use. Remove the low-salt agarose gel, from the side of the reservoir, surrounding the blocking salt gel used in forming this block.

11. Place the block with the prepared channels in the electrophoresis tank with the comb facing the cathode. Fill the tank with 1X THE buffer until the gel is covered. Carefully remove the comb. Filling the gel with buffer before removing the comb prevents the walls of the loading well from deforming and sticking to each other.

12. Pre-mix the DNA sample with 10x loading buffer (20% (w/v) Ficoll 400, 100 mM Tris-HCl (pH 8.0), 5 mM EDTA, 0.01% xylene cyanol FF) to a final concentration of 1-2 fold.

13. Carefully load the sample into the well and connect the electrodes to the power source.

14. Run the electrophoresis at a constant voltage of 100 V until the tracking dye (xylene cyanol) begins to migrate out of the gel. Alternatively, monitor the migration of DNA during the electrophoresis and stop the run when the DNA of interest reaches the end of the gel. Be careful that the target DNA does not begin to migrate out of the gel.

15. After turning off the power, remove and rinse the remaining working buffer in the channel.

16. Carefully return the agarose blocking gel to its original position in the canal.

17. Place the channel block back into the electrophoresis chamber. Fill the reservoir with 1x THE buffer until the buffer level matches the gel surface.

18. Continue electrophoresis at a constant voltage of 100 V for an additional 10 - 40 min to elute the gel-purified DNA into the sample collection reservoir.

19. Turn off the power and transfer the buffer with eluted DNA from the collection channel using a pipette into a clean microtube.

The essence and industrial applicability of the claimed group of inventions are explained by the following examples, without in any way diminishing the claims under the invention formula in all particular forms of embodiment of the features.

Example 1

Extraction of DNA from plant, animal, insect, fungal and microbial tissues. Protocol for direct purification of total DNA from a mixture of cells (tissue) using a lysis solution with SDS and proteinase K.

1. A homogenized piece of plant, animal or other tissue of no more than 100 μg, obtained by freezing in liquid nitrogen or at -80°C, and grinding in a cooled mortar or mixer, is placed in a 1.5 ml microtube. An alternative method of tissue homogenization is grinding the tissue with a pestle in a microtube at room temperature in the presence of a lysing solution.

2. Add 500 µg of 1% SDS solution (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0) to the test tube with homogenized tissue and add proteinase K (endopeptidase K; EC 3.4.21.64) to a final concentration of 100-200 µg/ml and mix well with a stirrer.

3. This mixture is incubated at 55°C for at least one hour.

4. Preliminary preparation of the sample for application to the channel. A portion of the sample in the lysis buffer, in a volume of 100 μl, is transferred to a new microtube containing 20 μl of 10x buffer for application to the gel with XyleneCyanol FF dye, and mixed well on a stirrer.

5. At this stage, the salt block (AS2) is temporarily removed from the channel so that the channel is filled with buffer only.

6. Apply no more than 50 µl of the prepared sample with the loading buffer to the loading slot. Start electrophoresis at a constant voltage of 100 V, during which time the entry of the DNA sample with the XyleneCyanol FF dye into the agarose block can be observed.

7. After the XyleneCyanol FF dye has begun to move freely along the channel, stop electrophoresis. Drain all the buffer from the channel and return the salt block (AS2) to the channel. Fill the elution space with electrophoresis buffer (in a volume of 1 ml, until the gel is completely covered) and continue electrophoresis at a constant voltage of 100 V for 10-20 minutes. Collect all the eluate from the channel containing DNA in a microtube.

8. The collected eluate will contain residues of the XyleneCyanol FF dye, which do not interfere with subsequent enzymatic reactions. But which can also be removed together with the salt residues from the DNA solution using ultrafiltration on Amicon Ultra-2 CentrifugalFilterUnit columns (Millipore) or the standard procedure of DNA precipitation in isopropanol.

As a result, 2 μg of DNA were obtained from 10 μg of tissue, which corresponds to 90% of the total DNA in plant tissue to the mass of tissue of young sprouts. DNA purity was measured spectrophotometrically for the presence of polysaccharides and polypeptides. Spectrometric data showed a complete absence of polypeptides in the sample and the presence of polysaccharide residues migrating into the sample from agarose. The ratio of the absorption values of nucleic acids at A ₂₆₀ (absorption at 260 nm) to the absorption value for polysaccharides at A ₂₃₀ (absorption at 230 nm) is at a value above 2. This corresponds to the minimum amount of agarose residues in the sample that came from the gel, and thus the amount of pure DNA exceeds 70-80%.

Example 2

Isolation and purification of total DNA from soil.

The protocol can also be used for other "complex" samples (e.g., feces) and tissues containing a high amount of polymer molecules, pigments, which cannot be separated by any other method. The scale in this example is taken for obtaining DNA from samples weighing 1-5 grams, but can be scaled to microvolume and sample mass.

Preliminary sample preparation

1. Soil samples are ground at -80°C, obtained by freezing in liquid nitrogen or at -80°C and grinding in a mortar or mixer cooled to -80°C.

2. A sample of homogenized soil weighing no more than 5 grams is placed in a 50 ml test tube, to which a CTAB solution (2% CTAB, 1.5 M NaCl, 10 mM Na ₃ EDTA, 0.1 M HEPES acid, pH 5.3) is added to a final volume of 40 ml. The mixture is stirred very vigorously on a stirrer and incubated overnight at 65°C.

3. Test tubes with soil samples are centrifuged to clarify and precipitate the soil sediment in a centrifuge for large test tubes, with cooling at +4°C, for 10 minutes at 10 thousand revolutions per minute.

4. The clarified supernatant solution is divided into two equal portions of 20 ml and transferred to new 50 ml test tubes, to which an equal volume of 100% isopropanol (20 ml), cooled to -20°C, has already been added and vigorously mixed on a stirrer.

5. The tubes are centrifuged at +4°C for 10 minutes at 10 thousand revolutions per minute, and all the supernatant is drained. A brown sediment of DNA containing soil components has formed at the bottom of the tube.

6. Add 10 ml of 70% ethanol to the test tubes with sediment to wash the sediment and mix well with a stirrer. Centrifuge the test tubes at +4°C for 3 minutes at 10 thousand revolutions per minute.

7. The supernatant alcohol solution is completely poured off, not dried, and 1 ml of 1x TE solution is immediately added to the sediment, mixed well and incubated at 65°C until the DNA sediment is completely dissolved. DNA solutions from both tubes are combined and the combined DNA solution can be stored in 2-5 ml microtubes.

Purification of DNA from a soil sample after precipitation and dissolution.

The next steps for cleaning the soil samples start with step 4 (Pre-treatment of the sample for application to the channel) corresponding to the DNA purification from Example 1.

As a result, 5 μg of highly purified and high-molecular DNA was obtained from a total mass of 10 μg of soil DNA obtained by precipitation with isopropanol, without purification. This corresponds to 50% of the total DNA mass, since most of the soil DNA consisted of fragments of different lengths, and DNA smaller than 10 thousand nucleotide pairs was not the purpose of purification and was discarded during purification. DNA purity was measured spectrophotometrically for the presence of polysaccharides and polypeptides. Spectrometric data showed a complete absence of polypeptides in the sample and the presence of polysaccharide residues migrating into the sample from agarose. The ratio of the absorption values of nucleic acids at A ₂₆₀ (absorption at 260 nm) to the absorption value for polysaccharides at A ₂₃₀ (absorption at 230 nm) is above 2. This corresponds to the minimum amount of agarose residues in the sample that came from the gel, and thus the amount of pure DNA exceeds 75%.

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46. Patent for utility model RU 84381 U1 dated 10.07.2009 (Institution of the Russian Academy of Sciences, V.A. Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences (IMB RAS) (RU)) "Device for automated extraction of nucleic acids from biological samples".

Claims (26)

1. A method for purifying and isolating high-molecular nucleic acids from a sample, comprising: - filling the electrophoresis channel zone with an agarose separating gel containing an electrophoresis buffer with low ionic strength; - application of a sample containing the target high-molecular nucleic acid to an agarose separating gel containing a low ionic strength electrophoresis buffer; - purification of high molecular weight nucleic acid by electrophoresis; - stopping electrophoresis after separation of high-molecular nucleic acid from contaminants until the purified nucleic acid exits the agarose separating gel containing electrophoresis buffer with low ionic strength; - installation of an agarose blocking gel with a salt content in the range of 0.5-1 M sodium chloride in the electrophoresis channel in the direction of movement of nucleic acids, after an agarose separating gel containing a buffer for electrophoresis with a low ionic strength, in which the purified nucleic acid is located, with a gap between the gels of at least 0.5 cm; - filling the gap between the gels with electrophoresis buffer of low ionic strength; - resumption of electrophoresis until the target nucleic acid migrates into the gap between the gels; - selection of purified high-molecular nucleic acid from the gap between the gels. 2. The method according to claim 1, wherein the collection of purified nucleic acid from the gap between the gels is carried out using an automatic pipette or a laboratory robot. 3. The method according to claim 1, wherein the purified nucleic acid is collected from the gap between the gels into a microtube for further use. 4. The method according to claim 1, wherein before purifying the high-molecular nucleic acid by electrophoresis, the duration of electrophoresis is determined. 5. The method of claim 1, wherein the low ionic strength electrophoresis buffer is 20 mM Tris and 20 mM HEPES. 6. The method according to claim 1, wherein the sample further comprises a mixture of biological molecules including non-target nucleic acids or short oligonucleotides, or proteins, or peptides, or polysaccharides, or lipids, or secondary metabolites, or humic substances, or pigments, or polyphenols. 7. A device for purifying and isolating high-molecular nucleic acids from a sample using the method of paragraph 1, comprising: - at least one electrophoresis channel; - a zone of the electrophoresis channel for filling with an agarose separating gel containing an electrophoresis buffer with a low ionic strength; - an electrophoresis channel zone for installing an agarose blocking gel with a salt content in the range of 0.5-1 M sodium chloride, - a gap between the zone for filling with an agarose separating gel containing a low ionic strength electrophoresis buffer and the zone for installing an agarose blocking gel with a salt content in the range of 0.5-1 M sodium chloride; - wherein the gap between the zone for filling with an agarose separating gel containing a buffer for electrophoresis with a low ionic strength and the zone for installing an agarose blocking gel is designed with the possibility of filling with a buffer for electrophoresis with a low ionic strength and is at least 0.5 cm. 8. The device according to item 7, in which the electrophoresis channel is made horizontal or vertical. 9. The device according to item 7, in which the electrophoresis channel is made open or closed. 10. The device of claim 7, wherein the low ionic strength electrophoresis buffer is 20 mM Tris and 20 mM HEPES. 11. The device according to item 7, made of plastic, transparent to visible and UV radiation. 12. The device according to item 7, which additionally includes electrodes for carrying out electrophoresis in the electrophoresis channel. 13. The device according to claim 7, which further includes a sensor for collecting information about the isolated high-molecular nucleic acids, connected to the information processing unit and the display unit.