RU2114175C1 - Method of nucleic acid cloning - Google Patents

Abstract

FIELD: biotechnology, molecular biology. SUBSTANCE: nucleic acids cloning is carried out by contacting nucleic acids with medium components in vitro containing cell-free enzyme system and substrates that are necessary for exponential multiplication of nucleic acids. Then liquid phase is immobilized by its placing in solid base having the developed surface at an average pore size from 100 mcm to 5 nm followed by incubation under conditions providing exponential multiplication of nucleic acids, detection and analysis of obtained nucleic acid colonies. EFFECT: improved method of nucleic acids cloning. 20 cl, 2 dwg

Description

 The invention relates to the field of molecular biology and biotechnology, and in particular to methods for nucleic acid cloning. An advantageous field of use is the production of individual nucleic acids.

 Currently, nucleic acid cloning is carried out in vivo, i.e. in living cells. The cloning procedure involves a number of steps, such as embedding (doping) nucleic acid fragments into cloning vectors (eg, plasmids or viral genomes), transforming living cells with recombinant molecules obtained, and obtaining transformed cell colonies or viral “plaques” on the surface of nutrient agar [1 ]. It uses the ability of living cells and viruses to exponentially multiply, so that you can quickly get large offspring of each transformed cell that carries the desired nucleic acid. This procedure is often called molecular cloning, which is not entirely true, since cells or viruses are cloned, not molecules. In addition, recombinant nucleic acids multiply in cells along with cellular DNA and RNA, which requires subsequent purification.

 Currently, there are a number of known methods for the exponential propagation of nucleic acids in cell-free systems, such as RNA propagation by viral RNA-dependent polymerases (GRP), DNA multiplication in the polymerase chain reaction (PCR), and isothermal three-enzyme system of self-sustaining reproduction (3CP). In vitro exponential propagation reactions of nucleic acids are carried out in liquid media containing components of a cell-free enzyme system, including a reaction buffer, corresponding enzymes, nucleotide substrates and, when necessary, polymerization seeds. Due to the exponential nature of the described propagation reactions, each of them could be used to quickly obtain numerous offspring of single nucleic acid molecules. This would make it possible to clone nucleic acids in vitro, that is, would provide an alternative to traditional gene cloning in living cells.

 In the GRP reaction, exponential synthesis is achieved due to the fact that both messenger RNA and synthesized RNA remain single-stranded, and both serve as equally efficient matrices in subsequent replication cycles. This reaction does not require seeds. Viral RNA polymerases, such as Qβ replicase, exhibit a narrow matrix specificity based on the recognition by the enzyme of special structures that are present in specific matrix RNAs but are absent in other RNAs. RNAs containing such structures are called replicating RNAs. Other RNAs that are not replicating can be propagated in the GRP reaction if they are integrated into the chain of replicating RNA [2]. Such recombinant replicating RNAs were used for extracellular reproduction of mRNAs encoding proteins [3].

 PCR is used for extracellular DNA reproduction. To carry out this reaction, two oligonucleotide seeds are necessary, which are complementary to sections of double-stranded DNA chains that limit the propagated fragment (target). These seeds are annealed with DNA and grow in mutually opposite directions by DNA polymerase. Unlike the GRP reaction, the matrix and synthesized DNA strands end up in a duplex, which must be melted at elevated temperature to allow subsequent replication cycles in which each of the strands serves as a matrix. The process is repeated many times by cyclically changing the temperatures at which annealing of the seed and melting of the DNA duplex occurs, which leads to the exponential propagation of the target DNA [4]. Currently, PCR is carried out using heat-resistant DNA polymerases that can withstand multiple temperature changes without loss of activity [5]. The need for cyclic temperature changes requires special equipment and makes PCR an order of magnitude slower than the GRP reaction. At the same time, any target DNA can be propagated by PCR in the presence of a pair of specific seeds, without the need for the manufacture of a recombinant molecule.

 The method of propagation of nucleic acids in a three-enzyme system of self-sustaining reproduction (3CP) combines the advantages of PCR and GRP reaction. 3СР is based on the combined action of three enzymes: DNA-dependent RNA polymerase, reverse transcriptase, and RNase H [6]. As a starting matrix, a double-stranded DNA fragment carrying a promoter of any RNA polymerase and single-stranded RNA can be used at each end. RNA polymerase (for example, T7 phage RNA polymerase) uses double-stranded DNA to synthesize multiple single-stranded RNA transcripts from the DNA sequence following the promoter on each DNA strand. Reverse transcriptase (e.g. reverse transcriptase of avian myeloblastosis virus) synthesizes double-stranded cDNA copies of RNA transcripts using seeds complementary to the 3'-ends of the transcripts and containing the RNA polymerase promoter sequence to restore this sequence at each end of the cDNA. RNase H destroys the RNA matrix involved in heteroduplex RNA / DNA after synthesis of the first cDNA strand, thereby allowing the second cDNA strand to be synthesized. The action of RNA polymerase and RNase H leads to the formation of single-stranded matrices, which allows exponentially multiply nucleic acids in the absence of cyclic temperature changes. The rate of 3CP reaction is comparable to the rate of GRP reaction, and like PCR, the use of 3CP does not require the creation of recombinant nucleic acids. The product of the 3CP reaction is a mixture of double-stranded DNA and single-stranded RNA molecules.

 As a prototype of the present invention can be considered the work of Levison and Spigelman [7], who stated that they were able to clone RNA molecules using Qβ replicase reproduction system. They observed RNA synthesis in about half of the samples after adding an RNA matrix diluted to such an extent that an average of 0.5 RNA molecules per sample was obtained. Obviously, the technique used cannot guarantee the progeny of a single molecule, since due to the random distribution in the sample, there may be two, three or more matrix molecules. Moreover, in these experiments, the synthesis product was not identified, and the authors' conclusion was questioned by the observation that RNA synthesis in the Qβ replicase system can occur even in the absence of an added matrix [8]. It has recently been shown that this spontaneous RNA synthesis is caused by replicating RNAs polluting the surrounding space, and that on average the sample contains up to 100 foreign replicating RNA molecules [9]. The contamination problem is also inherent in the PCR and ZSR reactions, although it is not as serious as in the case of the GRP reaction, since the multiplication of nucleic acids is controlled by oligonucleotide seeds complementary to the target. More significant in this case is the limited specificity of the seeds. Due to the presence of incorrect base pairing and heterogeneity of oligonucleotide seeds, it is possible to anneal the seeds on a foreign matrix present in the sample, which begins to compete noticeably with the correct annealing while lowering the proportion of specific matrices in the sample below a certain limit. At least 100 copies of the specific template must be present in the sample for reliable PCR start-up [10]. Thus, none of the existing methods of extracellular reproduction of nucleic acids allows one to achieve true in vitro cloning, that is, to obtain homogeneous offspring of a single molecule in a cell-free system.

 The present invention is based on our discovery of the fact that nucleic acids, like microorganisms, can be grown as colonies in an immobilized medium. The proposed method allows to clone nucleic acid molecules in vitro, despite the contamination of the sample with foreign matrices and non-specific annealing of seeds on foreign matrices. It is possible to use any acellular systems of exponential reproduction of nucleic acids, such as the GRP reaction, PCR, or 3CP reaction. In contrast to the prototype and other known methods of propagation of nucleic acids in liquid media, the offspring (clone) of each single nucleic acid molecule forms a colony of molecules in a limited area around the parent matrix. Different colonies occupy, as a rule, different zones of the immobilized medium, which makes it possible to observe individual colonies and to manipulate them separately.

 In FIG. 1 schematically depicts the growth of nucleic acid colonies in an immobilized medium; in FIG. 2 RNA colonies grown in a thin layer of immobilized medium containing Qβ components of the replicase system.

 The immobilized nucleic acid cloning medium comprises a liquid phase enclosed in a solid base. The solid base of the immobilized medium may have a diverse structure, for example, porous, fibrous, mesh, capillary, layered, folded. The main requirement for a solid base is the presence of developed surfaces that penetrate the liquid phase, the average distance between which provides a degree of immobilization of the liquid due to its friction against the surface and the structuring of water near the surface. It is preferable to use a solid base with an average distance between surfaces (pore size) from 100 μm (large-pore base) to 5 nm (fine-pore base). The upper limit of pore size should be less than the distance by which propagated nucleic acids are able to diffuse during the incubation period (of the order of 1 mm per hour of incubation at room temperature for nucleic acids with a length of about 100 nucleotides, cf. FIGS. 2A - 2B). The lower limit of pore size should be slightly larger than the linear dimensions of nucleic acids and / or enzymes to ensure their mobility necessary for the nucleic acid multiplication reaction to proceed. A three-dimensional network formed by individual polymer molecules or their aggregates can serve as a solid base; glued, sintered or pressed water-insoluble powders, fine granules or crystals; sponge materials; tightly packed fibers, capillaries or films (e.g. polymer film or metal foil). The main requirements for the material from which the solid base is made is its chemical inertness under incubation conditions, the hydrophilicity of the surface formed by it, and the absence of physical effects on the enzymes (for example, their irreversible sorption on the surface), which entail their inactivation. Suitable materials for the manufacture of a solid base are various organic or inorganic carriers used in biotechnology for chromatography and electrophoresis of biopolymers, for immobilization of enzymes, as well as for the growth of microorganisms, cells and viruses. Examples of such carriers are agarose, polyacrylamide, gelatin, alginate, carrageenan, cellulose, silica gel, sponge titanium, hydroxyapatite, chemically crosslinked agarose, dextran and polyethylene glycol, as well as combinations and derivatives thereof.

 According to the invention, the immobilized medium for cloning nucleic acids also contains components of a reproduction system comprising a cell-free enzyme system capable of exponentially multiplying nucleic acids. Examples of such a cell-free system are discussed above. The enzymes that make up the cell-free system can either be dissolved in the liquid phase or immobilized on a solid basis. The reaction buffer, propagated nucleic acids, substrates, and (where necessary) seeds are introduced into the liquid phase.

 When propagated in an immobilized medium, nucleic acids form colonies, as schematically shown in FIG. 1. The upper diagram shows a fragment 11 of an immobilized medium formed by a solid base network 12 and an aqueous solution 13 containing enzyme molecules 14, nucleic acid matrices 15, as well as substrates and reaction buffer. As a result of incubation of the immobilized medium for a certain time at a temperature suitable for the propagation of nucleic acids, colonies of nucleic acids 16 are formed in the localization zones of the parent matrices.

 Preferably, the solid base of the immobilized medium used for cloning can reversibly interact with nucleic acids. This allows you to significantly suppress the diffusion of nucleic acid molecules and to obtain smaller colonies with sharper edges and a higher concentration of nucleic acids; in other words, significantly increase the resolution of the method. Most hydrophilic polymers are capable of interacting with nucleic acids due to the formation of hydrogen bonds. This ability can be enhanced by chemically modifying the solid support with positively charged groups capable of ionic interactions with phosphate nucleic acid residues and / or moderately hydrophobic groups capable of interacting with purine or pyrimidine rings. For example, a solid base can be modified with weakly cationic groups, such as ethylaminoethyl or diethylaminoethyl, or intercalating dyes, such as ethidium or propidium. Intercalating dyes are capable of hydrophobic interactions with stacked nucleic acid bases and at the same time carry a positive charge. Moreover, the presence of intercalating dyes in an immobilized medium makes it possible to detect nucleic acid colonies using fluorescence (see below).

 To prevent the spread of nucleic acids in the immobilized medium during the growth of colonies, it is very important to prevent the presence of immobilized fluid. In particular, condensation of water on the surface of the immobilized medium cannot be allowed. At the same time, drying of the immobilized medium should not be allowed, which can lead to inactivation of enzymes. Drying of the immobilized medium can be prevented by incubating in a tightly closed chamber, cassette or sealed plastic bag, as well as wrapping the medium with a waterproof film or layering mineral oil.

Preferably, the propagation reaction does not start until the medium is completely immobilized. This condition is easily satisfied in the case of PCR, since in this case the reaction is triggered by a cyclic change in temperature. In the case of expression reactions or isothermal propagation, such as GRP or 3CP reaction, this condition is fulfilled either by preparing the medium at or around 0 ^o C, when the reaction rate is low, or by placing enzymes and substrates in different zones of the immobilized medium, after which the substrates diffuse to the enzyme zone, or by using protected substrates (chemically inaccessible to the enzyme), followed by deprotection and release of the normal substrate. An example of a protected substrate is the photosensitive ATP derivative, in which γ-phosphate is modified by a 1- (2-nitro) phenylethyl group [11], and which can be converted into ATP by photolysis to initiate the propagation reaction.

 For cloning, it is preferable to use an immobilized medium prepared in the form of a thin layer. In this case, the growing colonies are located in two dimensions, which facilitates their observation, analysis and reseeding, and also allows you to make replicas, for example, by partially transferring the colonies to a membrane filter. Replicas can be used to analyze colonies, to transfer them to a new medium, and also to store for a long time.

 The small thickness of the layer reduces the temperature gradients in the transverse direction (which is especially important in the case of PCR) and facilitates the diffusion of substrates into the enzyme layer when using sandwich media with separate enzyme and substrate layers (see below). Preferably, the layer thickness does not exceed the size of the colonies (which is determined mainly by the linear diffusion rate of nucleic acids), that is, within a few millimeters. It is most preferable to use very thin layers, as this increases the resolution of the method. The lower limit of the layer thickness depends on the mechanical strength of the solid base, on the ability to prevent drying out of the layer during manipulations and in the process of reproduction, as well as on the sensitivity of the method used to analyze colonies. In practice, satisfactory results are obtained with a layer thickness of 1 mm to 50 μm. Of course, thicker (e.g. 10 mm) and thinner (up to 1 μm) layers can be used. If thicker layers are used, it is preferable to place the enzymes and substrates in different layers that are stacked on top of each other, and / or introduce nucleic acid matrices on the surface of the layer or between layers in order to limit the propagation reaction to a narrow zone near the layer surfaces. Thinner layers are preferably immobilized on a substrate of a durable material, such as a glass, metal or plastic plate or film.

 The choice of thin layer preparation technique depends on the properties of the solid base, on the temperature of the reaction, on the properties of the enzymes used for the reaction (such as their ability to tolerate the formation of the polymer base), and whether a single-layer or multi-layer immobilized medium is used.

 Single-layer media can be conveniently used when all components of the enzyme system can be mixed without starting the reaction (as in the case of PCR). At the same time, single-layer media can also be used when it is preferable to keep the enzymes separate from the substrates until the medium is completely immobilized. In this case, the enzyme layer, which also contains nucleic acid matrices, is prepared without substrates and covered with a semipermeable (e.g., dialysis) membrane, or a solid base is used to prepare the enzyme layer, which can retain nucleic acids and enzymes (e.g., modified by positively charged groups). To start the propagation reaction, such a layer is brought into contact with a substrate solution.

 In many cases, it is preferable to use multilayer (sandwich) media in which different layers are brought into direct contact with each other at the right time. For example, enzymes and substrates can be separated into different layers, and the reaction is triggered by the contact of the layers due to the diffusion of the substrates into the enzyme layer. The use of sandwich media may also be preferable if the distribution of enzymes and substrates is not required, as, for example, in PCR. For example, the second layer may be a membrane filter used to analyze colonies onto which nucleic acids are transferred simultaneously with their synthesis. The second layer may also contain components of the expression system of nucleic acids and layered on the first layer at the end of PCR to analyze colonies for the expression product. In this case, a third layer can also be used, which can be a membrane filter for transferring expression products or a membrane filter impregnated with substrates of enzymes synthesized as a result of expression to analyze colonies by the activity of encoded enzymes.

 Thin layers can be made by various methods, such as impregnation of a dry solid base with solutions containing enzymes and / or substrates, or the inclusion of enzymes and / or substrates during gelation or synthesis (polymerization) of a solid base.

 Gelling solutions, such as those based on agarose, gelatin, carrageenan, are mixed with enzymes and / or substrates and poured, for example, into Petri dishes or between two even surfaces. In the latter case, it is possible to obtain very thin layers of the gel and to avoid the formation of a meniscus. Enzymes and / or substrates can also be included in the layer during the synthesis of a solid base, for example, during the polymerization of acrylamide [12] or photoinduced polymerization of ethylene glycol [13]. In cases where the formation of a solid base occurs under conditions that are too harsh for enzymes and / or substrates, a solid base is first prepared and then impregnated with an appropriate solution. For example, it is possible to increase the thermal stability of an agarose gel (so that it can be used in PCR) by crosslinking the agarose with epichlorohydrin or 2,3-dibromopropanol; a heat-resistant gel can also be obtained by crosslinking dextran with epichlorohydrin or N, N'-methylenebisacrylamide [14]. However, crosslinking occurs under conditions leading to the inactivation of enzymes and substrates of the propagation reaction. Therefore, a thin gel layer is first formed in the absence of enzymes and substrates and then impregnated with a solution containing enzymes and / or substrates. In the same way, polyacrylamide gel layers or a mixture of polyacrylamide and agarose can be prepared. Fibrous layers (e.g., based on cellulose or nylon) or porous layers (e.g., based on silica gel or sponge titanium) can be prepared simply by soaking the appropriate dry sheets, membrane filters or plates with a solution containing the components of the propagation system.

 Nucleic acids intended for cloning are introduced into the liquid phase of a solid medium by incorporation into an enzyme and / or substrate solution prior to its immobilization or by application to or between layers.

The propagation reaction is started by placing the immobilized medium in appropriate conditions: increasing the temperature of the medium, cyclic temperature change, exposure to light, bringing into contact the layers containing enzymes and substrates. The medium is incubated for a period of time, allowing the synthesis products to accumulate to a detectable level. For example, when multiplying nucleic acids, it is necessary that at least 10 ⁶ copies of nucleic acid are formed in each colony (that is, at least 20 replication cycles must occur, provided that the number of matrices doubles in each cycle), which corresponds to the lower limit of sensitivity of existing methods for detecting nucleic acids [15].

 At the end of the propagation reaction, nucleic acid colonies can be detected in various ways, such as staining with intercalating dyes (ethidium bromide, propidium iodide), autoradiography (if substrates labeled with a radioisotope are used for reproduction), and also with non-isotopic methods, such as using labeling of substrates with fluorescent groups and their modification with biotin or digoxygenin. Colonies can also be analyzed for any trait, such as the presence of a specific nucleotide sequence (by hybridization with a specific labeled probe) or the ability to encode a specific protein (by extracellular expression of the genes contained in nucleic acids). Often, detection of colonies is conveniently carried out after partial transfer to a membrane filter. In this case, the membrane filter is preferably applied to the growth layer before propagation, so that transfer occurs as the colonies grow. Detected clones can be further propagated using known in vitro nucleic acid propagation methods.

 The expression of nucleic acids for the analysis of colonies can be carried out in a liquid medium, however, it is preferable to carry out the expression in an immobilized medium formed in at least one thin layer. This allows you to simultaneously analyze a large number of colonies in one reaction. When expressed in an immobilized medium, expression products accumulate in those areas where colonies of the corresponding nucleic acids are present. When using isothermal propagation reactions (such as GRP or 3CP), the components of the expression system can be introduced into the same layer (or the same layers) as the components of the propagation system. If PCR is used for propagation, the expression system can be included in the same medium, but in a separate layer, which is layered at the end of PCR, or in a separate medium. In the latter case, DNA colonies are transferred to an expression medium, for example, using a membrane filter. For expression, nucleic acids are obtained in the form of RNA (as they are formed in the reactions of GRP or 3CP) and translated into proteins in a medium containing a cell-free translation system [16-18]. If nucleic acids are obtained in the form of DNA (as during multiplication in PCR), expression is carried out in a medium including a conjugate cell-free transcription / translation system [19-22]. The synthesized polypeptides can be identified in situ or on a replica filter in a variety of ways, such as using immunochemical reactions, or the ability of synthesized proteins to carry out specific enzymatic reactions or to bind specific ligands.

 The method of in vitro nucleic acid cloning can be used for the same purposes as in vivo cloning [1]. At the same time, it has several advantages: in vitro cloning is faster; it allows you to get a clone of any nucleic acid without any restrictions imposed by the cell, and allows you to get genes that are free from contamination of chromosomal DNA or cellular RNA. The obtained nucleic acid clones can be used for cell transformation, for therapeutic purposes (for example, for treatment using antisense RNA or DNA), or for use as matrices in protein synthesizing bioreactors [23].

 Due to the extreme sensitivity of the method, it is important to minimize the possibility of contamination of the sample with extraneous nucleic acids to the propagation stage. In this regard, it is preferable to carry out the stages preceding reproduction and the subsequent stages (analysis of colonies) in different laboratories. It is preferable to grow colonies in thin layers placed in sealed cassettes, which are opened only in the analytical laboratory.

 The proposed method is illustrated by the following examples of its implementation.

 Example 1. The cultivation of RNA colonies using the GRP reaction.

This example illustrates a method for propagating RNA in a thin layer of immobilized medium using RQ RNAs as natural Qβ replicase matrices. Powder low-temperature agarose (ultra-low gelling temperature agarose type IX, Sigma Chemical Company) is melted by heating in autoclaved buffer A (80 mm Tris-HCl pH 7.8, 2 mm MgCl ₂ , 1 mm EDTA, 20% glycerol), cooled to ≈40 ^o C and thoroughly mixed on a rotary shaker with a concentrated solution of Qβ replicase, purified as described by Blumenthal [24]. The final concentrations of agarose and Qβ replicase are 2% and 35 μg / ml, respectively. Then prepare a sandwich medium using one of the methods described below. When necessary, dilutions of messenger RNA are introduced into the substrate layer or between the enzyme and substrate layers in the form of a small aliquot.

(a) 1.5 ml of the above solution is layered on a 1% agarose layer containing substrates (1 mM ATP, GTP, CTP and UTP each) and buffer B (80 mM Tris-HCl pH 7.8, 25 mM MgCl ₂ , 2 mM EDTA, 10% glycerol), and prepared in 35 mm plastic Petri dishes. After pouring each layer, the cup is kept on ice to solidify the agarose. To multiply the RNA, the plate is incubated at room temperature or at 7 ^° C for 1 hour (on a thermostated table). RNA colonies show staining with ethidium bromide solution, as shown in FIG. 2, AB. In FIG. 2A shows the "spontaneous" growth of RNA colonies, observed when the upper (enzyme) layer is poured immediately after the solidification of the substrate layer. In FIG. 2B, a large number of colonies are visible that grew after a plate with a substrate layer was left open in the laboratory for 1 hour before pouring the enzyme layer. This experiment demonstrates the effectiveness of the proposed method in preventing the spread of "noise" growth throughout the reaction volume, as well as the potential danger from external (in particular, air) contaminants if nucleic acids are multiplied in the traditional way in a liquid medium. The dye can be included in the substrate layer when it is poured; in this case, RNA colonies can be observed during their growth (Fig. 2, B).

(b) 130 μl of a solution containing an enzyme and agarose is poured between a coverslip used in microscopy and a larger plastic plate, between which there is a gap of 0.4 mm. The coverslip was carefully removed and a nylon membrane filter impregnated with a substrate solution (4 mM ATP, GTP, CTP and UTP in buffer B) was applied to the agarose and dried. To detect RNA colonies, a [[α- ³² P] -] -labeled nucleotide is added to the substrate solution. After incubation of the medium for 20–40 min, the membrane filter is removed from agarose and washed from unincorporated substrates to propagate RNA. If reproduction was carried out in the presence of a labeled substrate, a membrane filter radioautograph is done. In FIG. 2, D shows the negative of the autograph of the original (substrate) membrane filter, and in FIG. 2, D is the negative of the radio-autograph of the filter-replica. The colonies in FIG. 2, D and 2, D are much less diffuse than in FIG. 2, A-B. The difference is explained by the presence in the nylon membrane filter of positively charged and hydrophobic groups capable of interacting with RNA. In the absence of a labeled substrate, colonies can be detected by staining agarose with ethidium bromide, as described above, or by hybridizing a membrane filter with specific probes, as described below.

RNA is sewn to the membrane filter under the influence of ultraviolet radiation and hybridized with a radiolabeled probe by incubation in a sealed plastic bag between sheets of filter paper [25]. The hybridization temperature is determined by the well-known formula [26] and optimized in preliminary experiments. After hybridization, the membrane filter is washed from the non-hybridized probe and a radio autograph is made. To re-hybridize the same membrane filter with another probe, the first probe is washed by boiling the membrane filter in a 0.1% Na-dodecyl sulfate solution. In FIG. 2, E and 2, G show the negatives of radio autographs obtained by sequential hybridization of the same membrane filter with radioactive-labeled probes specific for RQ87 _-3 RNA and RQ135 _-1 RNA, respectively. A mixture of these RNAs was introduced into the substrate layer in an amount of about 100 copies of each of them. This experiment demonstrates that different colonies contain different types of RNA and that the number of colonies corresponds to the number of matrices added. In other words, there is true cloning of RNA molecules.

 Example 2. The cultivation of colonies of nucleic acids using 3CP reaction.

A solution of low-temperature agarose in a buffer is mixed with enzymes and nucleic acid annealed with oligonucleotide seeds at 65 ^° C, and the enzyme layer is filled in as described in Example 1 (b). As a target, RNA, DNA or a mixture thereof is used. Use the reaction buffer, enzymes, seeds and incubation conditions according to the recommendations of Guatelli et al. [6]. A nylon membrane filter impregnated with a substrate solution containing 4 mM dATP, dGTP, dCTP, dTTp and 16 mm ATP, GTP, CTP, UTP in the reaction buffer is layered on the enzyme layer. After incubation, colonies of nucleic acids are identified by hybridization of a membrane filter with a labeled probe complementary to the inner region of the target, as described in example 1.

 Example 3. The cultivation of DNA colonies using PCR.

 All reaction components, including buffer, heat-resistant DNA polymerase, DNA sample, seeds and substrates, are mixed with a degassed solution of monomers (a mixture of acrylamide and N, N'-methylenebisacrylamide) and polymerization catalysts (ammonium persulfate and N, N, N ', N '-tetramethylenediamine) and fill in a thin layer of gel. Upon completion of the polymerization, a nylon membrane filter soaked in a reaction buffer is layered on the gel, wrapped with a heat-resistant film and the gel is placed on a metal thermostatic table, the temperature of which is controlled in the range required for PCR by an automatic device that includes two [27] or three [28] water thermostats and connected by hoses to a thermostatic table. Use the reaction components and PCR conditions recommended by Seikai et al. [5]. After 20 to 35 temperature cycles, DNA colonies are detected as described above. If RNA is used as a sample, it is first converted to cDNA [1].

 Example 4. Isolation of individual clones from complex mixtures of nucleic acids.

 This example describes a procedure based on the use of PCR. Of course, 3CP and GRP reactions can also be used. When using GRP, nucleic acids are converted into the form of replicating RNA by embedding the cloned sequences in the RQ RNA molecule [2].

 In conventional PCR, the desired nucleotide sequence from a mixture of nucleic acids can be selectively propagated by using two unique oligonucleotide seeds specific to this sequence, which necessitates the synthesis of a new pair of oligonucleotides to isolate each new sequence. The proposed method allows you to clone any nucleotide sequence using the same pair of universal seeds. The cloning procedure involves obtaining a mixture of nucleic acids whose size does not exceed the size that can be propagated in PCR (that is, up to 10 kb); hanging on them universal ends; and the use for their propagation of a pair of seeds complementary to the universal ends. In this case, each nucleic acid present in the mixture can be propagated and form a separate colony during PCR in a thin layer of immobilized medium. The next step is to identify the colony (or colonies) containing the offspring of a particular nucleic acid, and select this colony for further propagation and use.

 An initial mixture of nucleic acids can be, for example, a set of fragments obtained by digesting genomic DNA with any restriction enzyme [29], or a mixture of cDNA obtained from cytoplasmic mRNA [30]. Universal ends are hung on the fragments either by doping the fragments with oligonucleotides or by building up homopolynucleotide sequences. Preferably, the sequences introduced at the 3 'and 5' ends of the fragments are different from each other and are not complementary to each other. This can be achieved, for example, by lengthening the 3 'ends by doping dunnivorous DNA fragments with 5'-phosphorylated oligodeoxyribo nucleotide adapters compatible with the ends of the fragments, or by adding a homopolynucleotide using deoxynucleotidyl transferase [1], and lengthening 5' ends due to doping with oligoribonucleotides [31].

 After the propagation of the fragments in a thin layer of immobilized medium to obtain single colonies as described in example 3, the colonies are analyzed for the presence of a specific sequence (gene) by an appropriate method. This can be either hybridization with a specific probe complementary to the inner region of the gene (see above) if at least a partial nucleotide sequence of the gene or its homologue from another source is known, or if the gene is detected by its expression products in an in vitro transcription / translation system.

 Example 5. Analysis of clones of nucleic acids by expression in a thin layer of immobilized medium.

 If cloning is carried out using PCR, then the sequence of the RNA polymerase promoter, such as SP6 or T7 polymerase, is included at one of the universal ends introduced into the cloned fragments, as described in Example 4. The grown colonies are brought into contact with the expression layer. This is achieved either by layering the expression layer over the layer in which the PCR was performed, or by transferring the colonies to the expression layer using a membrane filter. An expression layer is prepared as described in Example 1, with the difference that instead of the components of the Qβ replicase system, the components of a suitable cell-free translation / transcription system are introduced into agarose [19-22]. If an isothermal propagation system is used for cloning, then the components of a suitable cell-free expression system [16-22] can be introduced into the same layer as the components of the propagation system. Expression products are identified in situ by their ability to carry out certain enzymatic reactions or by their ability to bind certain ligands or antibodies.

For example, clones carrying the phosphatase gene can be detected using a membrane filter moistened with a solution of 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium [32]. In those places where phosphatase was synthesized, purple staining appears. Genes encoding photoproteins, such as apobelin from Obelia geniculata, can be detected by luminescence [33]. In this case, the prosthetic group of the colenterazine protein is introduced into the expression layer embedded on a transparent substrate. After incubation of the expression layer for protein synthesis, it is placed with a substrate on a photographic film, and on the other hand, a membrane filter moistened with a Ca ⁺⁺ solution is layered on it. By diffusing into the expression layer and binding to obelin, Ca ⁺⁺ ions induce luminescence and, consequently, the illumination of the film in those places where apobelin was synthesized.

 Expression products can also be identified using a variety of immunochemical methods. Very sensitive methods based on chemiluminescent reactions catalyzed by alkaline phosphatase and horseradish peroxidase are most preferred. Suitable kits may be used, sold respectively by United States Biochemical Corporation and Amersham International plc.

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Claims (20)

 1. A method for cloning nucleic acids, comprising contacting in vitro nucleic acids with components of a medium containing a cell-free enzyme system and the substrates necessary for the exponential propagation of nucleic acids, incubation under conditions that ensure exponential propagation of nucleic acids, characterized in that the liquid phase of the medium before incubation they are immobilized by enclosing it in a solid base having a developed surface with an average pore size of from 100 μm to 5 nm, and detection and analysis are carried out from the obtained nucleic acid colonies.  2. The method according to p. 1, characterized in that the solid base has a porous, fibrous, mesh, capillary, folded or layered structure.  3. The method according to p. 1, characterized in that the solid base includes a gelling polymer substance.  4. The method according to p. 3, characterized in that the gelling polymer substance is selected from the group of substances, including agarose, polyacrylamide, their derivatives or a mixture thereof.  5. The method according to p. 1, characterized in that they use a solid base capable of reversible interaction with nucleic acids.  6. The method according to p. 5, characterized in that they use a solid base chemically modified by positively charged and / or hydrophobic groups.  7. The method according to p. 1, characterized in that at least one of the enzymes that make up the cell-free system is immobilized on a solid basis.  8. The method according to p. 1, characterized in that when immobilizing the liquid phase, the medium is shaped into at least one thin layer.  9. The method according to p. 1, characterized in that the medium is shaped into at least two thin layers superimposed on each other (the shape of a “sandwich”).  10. The method according to p. 7, characterized in that for the preparation of at least one thin layer using a membrane filter.  11. The method according to p. 1, characterized in that the medium is covered with a semi-permeable membrane that separates it from a solution containing a substrate.  12. The method according to p. 1, characterized in that the stages preceding the immobilization of the medium are carried out under conditions that prevent the multiplication of these nucleic acids.  13. The method according to p. 12, characterized in that the reproduction of nucleic acids is suppressed by spacing the enzymes and substrates that make up the reproduction system in different zones of the environment.  14. The method according to p. 1, characterized in that they use a cell-free reproduction system, including RNA-dependent RNA polymerase and ribonucleotide substrates, and nucleic acid intended for propagation, receive in the form of a replicating RNA.  15. The method according to p. 1, characterized in that the use of a cell-free reproduction system, including DNA-dependent RNA polymerase, reverse transcriptase, RNase H, ribonucleotide and deoxyribonucleotide substrates, and to the nucleic acid intended for propagation, add seed, complementary terminal areas of the specified nucleic acid or its fragment and including the nucleotide sequence of the promoter of the specified RNA polymerase.  16. The method according to p. 1, characterized in that for the preparation of the medium using a heat-resistant solid base and cell-free reproduction system, including heat-resistant DNA-dependent DNA polymerase and deoxyribonucleotide substrates, the nucleic acid intended for propagation is obtained in the form of DNA, to which add seeds complementary to the terminal sections of the indicated DNA or its fragment, and to carry out the propagation reaction, cyclic changes in the temperature of the medium between the melting temperature of DNA and the temperature A teratum conducive to annealing the indicated seeds with chains of the indicated DNA.  17. The method according to p. 1, characterized in that the detection and analysis of nucleic acid colonies is carried out in a thin layer of the medium.  18. The method according to p. 17, characterized in that the analysis of the colonies is carried out by expression of nucleic acids in a medium including a cell-free enzyme system capable of expressing nucleic acids, the liquid phase of the medium is immobilized by its conclusion in a solid base having a developed surface and having a porous , fibrous, mesh, capillary, folded or layered structure with an average pore size of from 100 μm to 5 nm, and identification by expression products.  19. The method according to p. 18, characterized in that the cell-free expression system is present in the same layer of the immobilized medium in which the nucleic acids are cloned.  20. The method according to p. 18, characterized in that the expression products are identified using a reaction selected from the group comprising a specific enzymatic reaction, binding of a specific ligand and a specific immunochemical reaction.

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