RU2616279C1 - Method for production of marker ladders for gel electrophoretic determination of nucleic acid fragment sizes - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for producing a special type of consumables for biomedical research, length markers of nucleic acid fragments (DNA and RNA marker ladders). The invention consists in testing of DNA and RNA fragments, constituting the ladder via amplification reaction by a "ring rolling" in rolling circle amplification embodiment. As amplification matrix, a single-stranded DNA ring molecule of desired size L is used (where L is the number of nucleotides), which is formed from a synthetic oligonucleotide of size L by intramolecular ligation. Ring ligation product is used as a matrix for annealing and elongation of "seed" primer 1, and primer 2 present in the reaction mixture provides for amplification starting according to amplification type. For polymerization reaction of polynucleotide chain, DNA or RNA polymerases are used having chain displacing activity, amplification is carried out in an isothermal or a quasi-isothermal mode. When adding to the reaction mixture of small amounts of restrictases and nickases, growing chains splitting may be achieved according to recognition sites for these enzymes, thereby reducing the amount of very extended amplification products in favour of shorter. To produce RNA marker ladders, used deoxyribo- ribonucleoside triphosphates and the mixture of DNA and RNA polymerases are used. To produce marker ladders of fragment sizes not multiple of the size of the ring matrix, primers with 5'-terminal sequence of any desired length and non-complementary annular original matrix are used. Another way to produce marker ladders woth the size of fragments not multiple of the size of the ring matrix is a simple mixture of products obtained by amplification of ring matrices of different sizes. The proposed method allows to obtain marker ladders in a significant amount with any size increments. They can be used in medical diagnostic laboratories, testing laboratories and research and development laboratories during implementation of all types of all gel electrophoretic analysis of nucleic acids.

EFFECT: use of marker ladders produced in the manner described above, is identical to that for all commercially available marker ladders.

5 cl, 5 dwg, 9 ex

Description

The invention relates to biotechnology and molecular biology and is associated with the production of a special type of consumables - markers of the lengths of fragments of nucleic acids. They can be used by medical diagnostic laboratories, examination laboratories and research laboratories for all types of gel electrophoretic analysis of nucleic acids. The essence of the invention lies in a new method for producing marker ladders, which consists in producing DNA or RNA fragments that make up a ladder using the rolling ring amplification reaction (ACC) in a variant of ramification using a synthetic ring matrix of a given size. The proposed method allows to obtain marker stairs in a significant amount with any size step in a wide range.

Description of drawings

FIG. 1. Scheme for the preparation of a ring matrix and amplification by a "rolling ring" in the variant of ramification (L is the size of the matrix in nucleotides).

FIG. 2. Electrophoregram of marker ladders: M - commercial marker, 1 - marker ladder obtained using a ring matrix of 50 nucleotides, 2 - marker ladder obtained using a ring matrix of 60 nucleotides, 3 - marker ladder obtained using a ring matrix of size 100 nucleotides.

FIG. 3. The scheme of the amplification of the "rolling ring" in the variant of ramification, combined with endonuclease cleavage.

FIG. 4. The scheme of the amplification of the "rolling ring" in the version of the ramification that occurs when receiving the RNA marker ladder.

FIG. 5. The scheme of the amplification of the "rolling ring" in the variant of ramification, proceeding upon receipt of marker stairs with a step different from the size of the ring matrix.

Description of the invention

The detection of restriction endonucleases and their use for the cleavage of DNA molecules into fragments of a certain length gave impetus to the electrophoretic analysis of nucleic acids in polyacrylamide and agarose gels. To compare the results of different electrophoretic separations between themselves, it was necessary to use marker molecules of known length, correlation with which by electrophoretic mobility made it possible to more accurately determine the sizes of DNA fragments formed by restriction enzymes. Among the first marker molecules were DNA phages and plasmids with specific sizes of restriction enzymes or with known nucleotide sequences. The most widely used were lambda phage and plasmid pBR322. Before the full sequence of lambda phage became known, the size of its restriction enzyme fragments was previously estimated using electron microscopy [Murray K., Murray N., 1975; Southern, 1979]. Chemically synthesized DNAs were used to determine the size of small fragments [Maniatis et al., 1975]. Plasmid vector pBR322, widely used for molecular cloning, after complete sequencing [Sutcliffe, 1978; 1979] began to be used as a marker of molecular masses of small fragments of DNA.

The book Gel Electrophoresis on Nucleic Acids, published in the Practical Approaches in Biochemistry Series, in the section on DNA gel electrophoresis [Sealey, Southern, 1982], recommended the use of DNA from those phages and plasmids for which nucleotide sequences so that the size of the restriction fragments can be accurately calculated. Table 1 is given in Appendix 1 to the book [Minter, Sealey, 1982], in which for a number of phages and plasmids the sizes of DNA fragments formed under the action of various restriction endonucleases with nucleotide sequences determined by that time were indicated. In the catalogs of a number of companies in the early 80s of the last century, special positions began to appear - the products of cleavage by different restrictase enzymes (or their mixtures) of phage and plasmid DNA molecules, serving as markers for gel electrophoresis.

Another type of markers are DNA marker “stairs”, the “steps” of which are identical and relatively evenly located in the gel track, and the intensity of the luminescence of bands of different sizes, as a rule, is brought together. For the first time, a DNA marker ladder with steps of 123 bp began to supply the American company Bethesda Research Laboratories, in the catalog of which in 1983 in the section "Gel Markers: Nucleic Acids and Protein" (pp. 22-25), along with markers from restriction enzyme fragments of lambda phage DNA, an innovative product appeared - 123 bp Ladder. A year later, the same company introduced the kilobase ladder to the market.

The beginning of the use of DNA marker ladders was laid by the work of American authors [Hartley, Gregori, 1981], in which they reported on the successful cloning of 34 repeats of a short section of the rat prolactin gene. The authors do not mention the use of cloned short repeats as marker ladders. However, when looking at electrophoregrams, the article raises the idea of the possibility of such an application, and it was this recombinant plasmid that became the source of the commercial product 123 bp Ladder [Hartley, Gregori, 1983]. A similar approach was patented in 1998 [Hyman, 1998]. Its main difference was the presence of an additional restriction enzyme site located at some distance from the same sites restricting repeats of 100 bp, which allowed us to exclude the amorphous band belonging to the residues of the vector with adjacent repeating regions in the upper part of the gel. A more complex plasmid containing several cassettes of short repeats 10 bp long, when cleaved with EcoRV restriction enzyme under conditions of underreduction, 10 bp were formed. DNA marker ladder designed by the authors [Hu et al., 2005].

In 2012, Vietnamese authors created a recombinant plasmid containing 8 repeats of 100 bp each. [Lan et al., 2012]. Having retreated 100 bp from each edge of this multicast, they selected primers for plasmid sequences, which upon amplification gave a product of 1000 bp in size. Its incomplete cleavage with the restriction enzyme SmaI led to the appearance of a ladder of bands with steps of 100 bp. Taking advantage of the fact that the genomic DNA of the flour crust Tenebrio molitor carries high-copy repeats (tens of millions of copies), the monomeric unit of which with the EcoRI restriction enzyme site within the repeat is 142 bp, Israeli authors [Barvish et al., 2007] proposed to create a marker ladder DNA to carry out by this enzyme undersplitting of total DNA of this insect. As a result, a characteristic staircase of stripes was formed.

In 2010, Chinese authors [Ye et al., 2010] created a different genetic engineering construct in which the insert cloned in several steps consisted of 15 repetitions, but each repetition was larger than the previous one by a monomer unit of 100 bp. Upon cleavage of the created recombinant plasmid with a total size of 8700 bp restriction enzyme EcoRI under conditions of complete restriction formed a set of fragments from 100 to 1500 bp. Somewhat earlier, other Chinese authors [Dongyi et al., 2008] created two recombinant plasmids in which they cloned amplified sections of lambda phage. With the restriction enzyme EcoRI cleavage of the two plasmids pYO and pYE created as a result, marker stairs with steps were formed: 300, 500, 700, 900, 1500, 3000 bp and 400, 600, 800, 1000, 1400, 2000 and 3000 bp respectively, where the upper bands are 3 kb in size. in both cases belonged to a vector plasmid. A year later, a similar work was published [Chen et al., 2009], with the only difference being that the recombinant plasmids created by cleavage produced larger DNA bands of 500, 750, 1000, 2000 and 3000 bp, forming a marker ladder of a different destination. In their next paper [Wu, Ye, 2011], these authors described new recombinant plasmids carrying a set of inserts, but the general principle of marker ladder generation remained the same.

The literature also reports on the creation of marker ladders by cloning in different plasmids individually DNA fragments of 200, 400, 800 and 1600 bp in size. [Planken et al., 2005]. However, to use such a marker ladder, the authors had to carry out restriction enzyme digestion of all plasmids, then carry out preparative gel electrophoresis followed by elution of the desired fragments, which, after purification, were mixed in the desired proportion.

There is one application and a series of US patents issued to various American companies that describe the creation of genetic engineering constructs containing in one plasmid different fragments (100, 200, 400, 800, 1200 and 2000 bp) fragments that can be split single restriction enzyme [Hartley, 1998, 2004, 2006, 2010, 2011].

In addition to restriction endonucleases, DNA marker ladders can be made using DNA ligases. Multiple (controlled) ligation of double-stranded DNA fragments with the formation of a mixture of fragments of different sizes serves as a convenient way to obtain DNA markers of the relative length of DNA molecules of a relatively small size. This technology for creating marker stairs is described in a US patent using an example of ligation of a DNA fragment of 20 bp in size, which ensures the formation of steps up to 1000 bp [Singer, 1998]. Based on this principle, many marker staircases with steps from 5 to 100 bp are currently being produced.

Abdel-Fattakh et al. 11 primers were selected (one direct and ten reverse), annealed at different distances on the plasmid vector pET-17 and providing the production of amplicons with sizes of 100, 198, 304, 399, 500, 750, 1000, 2000 and 2500 bp [Abdel-Fattakh, Gaballa, 2006]. They made up the DNA marker ladder with steps increasing upward. Indian authors [Chandrasekhar et al., 2008] to create a marker 100 bp stairs in the range from 100 to 1000 bp We selected one direct and only 5 reverse primers, which were annealed on two plasmids, one of which was a blank vector, and the other contained an insert of 504 (500) bp in size, which allowed amplification of both plasmids to form all steps on such a ladder 100 bp no passes. Authors from Taiwan [Chang et al., 2008] described a slightly different approach: ten different clones carrying inserts from 0 to 900 bp were amplified with one pair of primers selected for the flanking polylinker regions of the pGEM-T Easy plasmid. production of amplicons with sizes of 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 bp Iranian authors [Saidijam et al., 2011; Mostaan et al., 2015] also created a series of 11 recombinant plasmids containing inserts measuring 102 bp in size. up to 1398 bp, which, together with the single plasmid pTZR57R using PCR with one pair of primers, was used to generate "steps" of the ladder from 100 to 1500 bp For the formation of DNA bands of 6, 8 and 10 kbp they linearized suitable recombinant plasmids. The pGEM-T Easy vector, which does not contain inserts to create a marker ladder, was used by Thai authors [Riyajan et al., 2011]. Using combinations of seven forward and reverse primers, they succeeded in forming steps of 150, 300, 375, 500, 700, 1000, 1200 and 1625 bp. from the sequence of the vector itself.

In 2010, another group of Indian authors [Gopalakrishnan, Sellappa, 2010] using multiplex PCR created a multi-step marker ladder using lambda phage DNA as a template. For the annealing sites of one direct and 6 reverse primers, they selected a phage site at positions from 1216 to 2136 bp, eventually obtaining a marker ladder with steps corresponding to 100, 200, 400, 600, 800 and 1000 bp. Chinese researchers [Wang et al., 2010; 2011] created a similar marker 100 bp a staircase having the same steps from 100 to 1000 bp Lambda phage DNA was also selected by these authors; only the region amplified using ten forward and one reverse primers was located at positions between 6631 and 7630 bp. Almost the same portion of the phage lambda (7131-7630 bp) was used to amplify five fragments of 100, 200, 300, 400 and 500 bp. [Dawson, 1998].

Marker stairs with even larger steps were constructed by another Chinese authors [Zhang et al., 2012]. So, they selected primer pairs limiting lambda phage fragments of 1, 2, 3, and 4 kb during amplification. The larger fragments in their staircase (5, 6, 8, and 10 kb) were linearized recombinant plasmids carrying inserts of appropriate sizes. Marker stairs with small steps measuring just 6 bp developed by Spanish authors [Amills et al., 1996]. They selected 9 primers (5 straight, annealed with a shift of 6 bp, and 4 reverse, also annealed with a shift, but by 30 bp), which ultimately ensured the creation of a ladder with strip sizes from 90 to 204 bp

To carry out various options for pulsating gel electrophoresis, large marker stairs based on lambda phage, having a length of 48.5 kb, also exist. The handling of such extended DNA molecules requires special care, and in order to avoid their impulses, such markers are enclosed in agarose blocks even at the preparation stage, which are then placed in the wells of the gel. Thus, the creation by heating and cooling of marker ladders in the form of ordinary concatemers of the lambda phage [Waterbury, Lane, 1987] and the stitching of sequences of this phage by donating DNA with a T4 phage ligase followed by restriction by suitable restriction enzymes under conditions of incomplete cleavage [Cooney, 1992], covering a range of lengths from 48.5 to 1200 kb and from 48.5 to 200 kb respectively. When performing pulsating gel electrophoresis for use as a marker ladder, it was proposed to create multimers of plasmid DNA with sizes from 12 to 170 kb. [Hanlon et al., 1989]. Allelic ladders used in DNA identification using STR loci should be considered as another type of marker ladders [Puers et al., 1993; 1994; Schumm, Sprecher, 2006; Green et a., 2013].

Chang et al., 2008, speaks of the fundamental possibility of generating in vitro transcription under the influence of suitable phage RNA polymerases of marker RNA ladders with the same dimensions. It was also shown that using transcription using phage RNA polymerase to generate RNA molecules of different lengths for their use as length markers in gel electrophoresis, one insertion vector can be dispensed with if it is linearized by various restriction endonucleases whose sites are located on different distances from the start of transcription [Liggit et al., 1994; Audic et al., 1997].

An alternative to the complex construction of recombinant plasmids is the transcription of a rolling ring type using a special DNA ring matrix, which can have different sizes and can be prepared in one way or another. It has been shown that under the action of T7 RNA polymerase or RNA polymerase of E. coli, RNA molecules with a length of more than 7.5 thousand nucleotides can be produced, which are concatemer repeats, the monomeric unit of which is equal in size to the original ring [Daubendiek, Kool, 1997; Diegelman et al., 1998]. The presence of sections encoding cis ribozymes in these rings leads to cleavage of the primary transcript into RNA fragments, which form a characteristic ladder of bands with identical steps in the gel electrophoresis picture. In the work of Chinese authors [Wang et al., 2014], in addition to ribozyme cleavage, RNA concatemers formed as a result of transcription by a rolling ring, it was also proposed to fragment using RNase H and a modified DNA fragment annealed in the right places of the primary transcript.

Currently, the catalogs of many companies specializing in the supply of reagents for molecular biology include numerous marker ladders carrying fragments of DNA or RNA of a wide size range made in different ways. Among them, the 123 bp Ladder marker staircase, which has become a classic, is delivered by several companies. A very large line of such markers is supplied by a group of companies belonging to the Thermo Fisher Scientific concern. It is these firms that own most of the patents that describe methods for manufacturing various marker DNA ladders. For example, marker ladders GeneRuler, O'GeneRuler, MassRuler, FastRuler, ZipRuler Express are mixtures of different DNA fragments, each of which is purified by chromatography. Due to the individual cleaning of each fragment, it is possible for the specialists of this company to manufacture NoLimits marker stairs with any (from the existing list) “custom-made” steps for interested users solving specific narrow-minded tasks. O'RangeRuler marker ladders contain sets of DNA molecules that are the result of incomplete (partial) ligation of blunt-ended DNA fragments with repeating units with sizes from 5 to 500 bp In addition to DNA ladders, this company also produces RiboRuler RNA ladders, which are a mixture of chromatographically purified transcripts in the form of 8 steps for High Range (from 100 to 6000 nucleotides) and 7 steps for Low Range (from 100 to 1000 nucleotides).

New England Biolabs, Inc. supplies almost two dozen different marker ladders for gel electrophoresis of both DNA and RNA. Moreover, the range of transcript lengths in RNA ladders is quite large, from 17 nucleotides (for microRNA detection) to 9000 nucleotides. Separately, it is worth mentioning the Reverse Mass DNA Ladder ladder, in which smaller fragments are represented in greater numbers than all the others, while for many marker stairs, the lower steps are extremely poorly visible in the gel. In addition to linear marker DNA molecules, New England Biolabs, Inc. supplies a ladder of 9 supercoiled plasmids with sizes from 2 to 10 kb For pulsating gel electrophoresis, among other markers, a ladder is sold, which is a concatemer of the phage lambda, covering a range of 50 kb. up to 1 million bp This company also owns the TriDye DNA Ladder trademark, which hides a product that represents a mixture of three leading dyes: xylencyanol, bromphenol blue and orange G, migrating in a standard 1% agarose gel next to 4 kb DNA fragments. , 300 bp and 50 bp respectively. TriDye DNA Ladder is not intended for precise determination of the size of shared fragments, but serves only as a guideline for the duration of gel electrophoresis, although at the dawn of electrophoretic DNA analysis, these dyes were recommended to be used to determine DNA lengths.

Thus, today there are many ways to obtain marker stairs, and the market offers a wide range of commercial products. However, most commercially available marker ladders are obtained predominantly by restriction enzyme digestion of phages and plasmids. The production method used determines the relatively high cost of such products. In addition, the size of the “steps” in such marker stairs depends on the restriction enzymes used and the location of their recognition sites.

The essence of the proposed method is as follows. Marker ladders are obtained using the rolling ring amplification reaction (ACA, eng. RCA -rolling circle amplification) in the form of a branched ACC, or ramification (eng. Ramification). The matrix is a single-stranded ring molecule of the required size L (where L is the number of nucleotides), which is formed from a synthetic L-size oligonucleotide by intramolecular ligation using T4 RNA ligase (Fig. 1, reaction 1) or any other ligase capable of intramolecular ligation of single stranded nucleic acids. During the reaction, mainly linear ligation products are formed, which are concatemer (repeating) structures that do not interfere with the preparation of marker stairs. A necessary condition for a high yield of the target ligation product (ring) is the presence in the reaction mixture of a sufficient amount (5-7%) of polyethylene glycol (PEG); it is most convenient to use PEG with an average molecular weight of 4000-6000 Da, but you can also use PEG with other molecular weights and other inert thickeners, for example, polyhydric alcohols (glycerin, etc.), oligosugar, etc. (example 1).

In the next step, the ring ligation product is used as a matrix for annealing and elongation of primer 1, which acts as a "primer" primer (Fig. 1, reaction 2), and the second primer (primer 2) present in the reaction mixture ensures the launch of ACC according to the type of ramification (Fig. 1, reaction 3). The presence of two primers ensures the production of double-stranded DNA fragments that are multiple in size to the ring matrix (i.e., nL, where n is the number of polymerase rotations in the ring). Visualization of the results of ramification by gel electrophoresis gives a characteristic ladder of bands (Fig. 2).

For the polymerization reaction of the polynucleotide chain, depending on the type of marker ladder (DNA or RNA) obtained, DNA or RNA polymerase having chain-displacing activity, for example, Bst group DNA polymerase (large fragment of Bst, or Bst exo ^- , Bst 2.0, Bst 2.0 WarmStart, Bst 3.0 (New England Biolabs)), Vent exo ^- , Deep Vent, 9 ° N _m , Bsm exo ^- , Aac, etc. and T7 RNA polymerase. In the case of heat-labile DNA polymerase ^{(Bst exo -, Bst 2.0,} Bst 2.0 WarmStart, Bst 3.0, Bsm exo -, Aac) amplification was carried out isothermally at a temperature close to the temperature of the maximum activity of the enzyme and primer annealing (Example 2). The introduction of the enzyme is carried out after preliminary denaturation and annealing of the primer, although this step is not mandatory and serves only to increase the yield and quality of amplification products. Thermostable DNA polymerase (Vent exo ^- , Deep Vent, 9 ° N _m ) is introduced directly into the reaction mixture, followed by a finite number of cycles of temperature change, and then the mixture is kept at a constant temperature (Example 3). Thermal cycling is necessary to create extended targets by denaturation of the initial and formed matrices and annealing of the primers, and keeping at a constant temperature to increase the yield of reaction products. In this case, it is possible to use only the oligonucleotide precursor of the ring matrix for amplification (Example 4), which will also provide the necessary fragments, but with less efficiency.

By adding a small amount of restrictase or nickase to the reaction mixture, one can achieve cleavage of the growing nucleic acid chains at recognition sites of these enzymes predefined in the nucleotide sequence of the ring matrix, thereby reducing the number of very extended amplification products in favor of shorter ones (Example 5). With the correct ratio of polymerase and endonuclease, the desired product profile and, accordingly, the pattern of bands in the marker ladder are achieved (Fig. 3).

To obtain RNA marker ladders, a DNA ring matrix, ribo- and deoxyribonucleoside triphosphates, and two polymerase-shifting polymerase RNA polymerase T7 and Bst 3.0 DNA polymerase (Example 6) are used. A necessary condition for amplification in this case is the presence in the primer 1 at the 5'end of the T7 nucleotide sequence of the promoter -TAATACGACTCACTATAG. Since T7 RNA polymerase builds the RNA chain along the DNA chain, when amplified by a rolling ring, it will produce only a single-stranded RNA product, and ramification (formation of a set of concatemer products) will not proceed despite the presence of the second primer. In this regard, it is necessary to introduce additional DNA polymerase Bst 3.0, which has a unique ability to build DNA chains both from DNA and RNA matrix. In this embodiment of the reaction, both DNA and RNA chains are simultaneously formed, which allows both enzymes to work non-stop due to the presence of constantly synthesized suitable matrices (Fig. 4). The result of the reaction is a set of fragments, which are two types of double-stranded structures: DNA-DNA (dsDNA) and DNA-RNA, and double-stranded RNA-RNA is not formed. With the correct quantitative ratio of enzymes and deoxyribo and ribonucleoside triphosphates of DNA-RNA hybrids make up the bulk of the molecules. To obtain the final RNA marker ladder, the drug is first purified from polymerases, primers and triphosphates, and then subjected to DNase I treatment, which is able to cleave dsDNA and DNA-RNA hybrids into a DNA part, and subsequent purification. As a result, a set of single-stranded RNA fragments is obtained, the sizes of which will not be multiples of the size of the initial matrix due to the presence of a 5 ′ “tail” sequence, which must be taken into account when designing the ring matrix. A variant is possible when the nucleotide sequence of the T7 RNA polymerase recognition site is specified in the structure of the ring matrix. In this case, primer 1 will not have a non-complementary 5'-terminal part, and the sizes of the RNA fragments will be a multiple of the size of the original matrix.

To obtain marker ladders with fragment sizes not multiple of the size of the ring matrix, primers with a 5'-terminal (“tail”) sequence of any desired length and a non-complementary initial ring matrix can be used (Fig. 5). 5 '- "Tail" sequence can contain only one of the primers or both. By varying its length, it is possible to create marker stairs with any step size up to a single nucleotide, given that during amplification, the sizes of the 5 'tail sequence are added to the length of the ring (Example 7). The fragment sizes are determined by the equation n⋅ (L + M + N), where L is the size of the ring matrix, M is the length of the 5'-terminal sequence of the first primer, N is the length of the 5'-terminal sequence of the second primer, an is the number of polymerase rotations around the ring.

Another way to obtain marker staircases with fragment sizes that are not multiple of the size of the ring matrix is to simply mix the preparations obtained by amplifying ring matrices of different sizes (Example 8). For example, if you take two marker stairs with different fragment lengths: one 50, 100, 150, 200, 250, 300, 350, etc., the second 75, 150, 225, 300, 375, etc., you get marker stairs with the following fragment lengths: 50, 75, 100, 150, 200, 225, 250, 300, 350, 375, etc.

The reaction mixtures obtained after ACC can be purified by phenol-chloroform extraction according to known protocols and / or using commercial kits designed to isolate DNA and RNA from reaction mixtures. The final preparations of marker stairs should be stored at -20 ° C; their holding at temperatures above + 40 ° C is unacceptable. The use of marker ladders obtained by the method described above for gel electrophoretic analysis of nucleic acids is identical to that for all commercially available marker ladders (Example 9).

The invention is illustrated by the following specific examples.

Example 1. Obtaining a single-stranded ring matrix.

A single-stranded ring matrix is obtained from linear synthetic oligodeoxyribonucleotide (ODN) of any desired length L. ODN is ligated using T4 RNA ligase, for example, by the following procedure. To 1 μl of a solution containing 0.1 O.E. ONE, add 2 μl of ligase buffer, 3.0 μl of PEG6000, 2 μl of 10 mm ATP, 5 units. Act. T4 RNA ligase or any other ligase capable of intramolecular) ligation of single-stranded nucleic acids, and the reaction mixture is brought up to 20 μl, incubated for 18 hours at a temperature of 8-15 ° C and then heated for 10 min at 75 ° C. The obtained ring matrix can be further used for amplification reactions without further purification or subjected to purification by any known methods.

Example 2. Obtaining DNA marker ladders in isothermal amplification mode.

Prepare 20-50 μl of the amplification reaction mixture containing 1 μl of a single-stranded ring matrix solution, 1-2 μl of each of primers 1 and 2 (concentration 2.0 O.E. / ml), 2-5 μl of a mixture of deoxynucleoside triphosphates (dNTP) with a concentration of 2.5 mm, 2-5 μl of the appropriate buffer for DNA polymerase. Use the following amplification protocol. First, the samples are heated for 3 min at 95 ° C (denaturation), then the temperature is gradually reduced to the calculated temperature for annealing the primers (optimally 50-60 ° C), after which 1-5 units are added. Act. one of the Bst DNA polymerases (Bst exo ^- , Bst 2.0, Bst 3.0) or any other thermolabile DNA polymerase with chain-shifting activity and the reaction mixture is kept for 2 hours at the annealing temperature of the primers. Then, amplification products are isolated by the widely used phenol-chloroform extraction method.

Example 3. Obtaining DNA marker ladders in the regime of quasi-isothermal amplification.

Prepare 20-50 μl of the amplification reaction mixture containing 1 μl of a single-stranded ring matrix solution, 1-2 μl of each of primers 1 and 2 (concentration 2.0 O.E. / ml), 2-5 μl of a 2.5 mM dNTP mixture , 1-5 units. Act. Vent exo DNA polymerase ^— or any other thermostable DNA polymerase with chain-shifting activity, 2-5 µl of the appropriate DNA polymerase buffer. Use the following amplification protocol. First, the samples are heated for 3 min at 95 ° С (denaturation), then 15-20 cycles of thermal cycling are carried out (95 ° С - 30 s, 50-60 ° С (depending on the calculated temperature of primer annealing) - 30 s, 75 ° С - 30 s), and then the reaction mixture is kept for 2 hours at annealing temperature of the primers. Then carry out the selection of amplification products analogously to example 2.

Example 4. Obtaining DNA marker ladders without the use of primers.

Prepare the reaction mixture for amplification according to examples 2 or 3, but characterized by the absence of primers and the presence of a synthetic oligonucleotide - the precursor of the ring matrix. The amplification and purification protocols described in Examples 2 and 3 are used.

Example 5. Obtaining DNA marker ladders, combined with endonuclease cleavage.

The reaction mixtures are prepared for amplification according to examples 2 or 3, but characterized by the presence in them of an additional buffer in the required concentration for any suitable restriction enzyme or any suitable nickase that cleaves DNA chains at recognition sites previously specified in the nucleotide sequence of the ring matrix. Use the amplification protocols described in example 2 or 3, but characterized in that the introduction of 1-5 units. Act. restrictase or nickase is carried out simultaneously with DNA polymerase. The selection of products is carried out analogously to example 2.

Example 6. Obtaining RNA marker ladders.

Prepare 20-50 μl of the amplification reaction mixture containing 1 μl of a single-stranded ring matrix solution, 1-2 μl of each of the primers (concentration 2.0 O.E. / ml) carrying the T7 RNA polymerase promoter, 3 μl of a mixture of ribonucleoside triphosphates (NTP) with a concentration of 2.5 mM, 1 μl of a mixture of deoxyribonucleoside triphosphates (dNTP) with a concentration of 2.5 mm, 3 μl of buffer for T7 RNA polymerase and 3 μl of buffer for DNA polymerase Bst 3.0. Use the following amplification protocol. First, the samples are heated for 3 min at 95 ° С (denaturation), then the temperature is gradually reduced to 40-45 ° С, after which 10 units are added. Act. T7 RNA polymerase, 1 unit Act. DNA polymerase Bst 3.0 and maintain the reaction mixture for 3.5-4.0 hours at 40-45 ° C. Next, purification of the amplification products is carried out using any reagents for RNA isolation, for example, Trizol. To the resulting preparation, add 2 units. Act. DNase I and 5 μl of the corresponding buffer based on 50 μl of the reaction mixture, incubated for 30 min at 37 ° C, then again cleaned using any reagents for RNA isolation.

Example 7. Obtaining marker stairs in increments other than the size of the annular matrix.

The preparation of marker ladders with a step different from the size of the ring matrix is carried out similarly to the methods described in examples 2-6, characterized by the presence in the reaction mixture of primers with a 5'-terminal sequence of any desired length and an incomplete initial ring matrix.

Example 8. Obtaining marker stairs with mixed lengths of fragments.

Obtaining marker ladders with mixed fragment lengths is carried out using simple mixing of the preparations obtained by amplification of ring matrices of different sizes according to examples 2-5.

Example 9. Conducting gel electrophoretic analysis of nucleic acids using marker ladders.

DNA electrophoresis (for example, when analyzing the results of amplification, restriction, ligation, etc.), as well as RNA (for example, after isolation of RNA from biological samples), is carried out using polyacrylamide, agarose or mixed gels in any standard buffer in vertical chambers or horizontal type. Gels are stained with any of the known methods for detecting nucleic acids in gels and visualized in appropriate instruments.

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Claims (5)

1. A method of producing markers of lengths of DNA fragments (DNA marker ladders), characterized in that the rolling ring amplification reaction is used to produce a ramification variant involving a synthetic ring DNA matrix of a given size, two primers and DNA bias polymerases activity, providing the formation of a set of concatemer DNA products of a certain length each, a multiple of the size of the original ring DNA matrix. 2. A method for producing RNA length markers (RNA marker ladders), characterized in that the rolling ring amplification reaction is used to obtain a ramification variant with a synthetic DNA ring matrix of a given size, two primers, one of which contains a T7 recognition site RNA polymerase, and mixtures of DNA polymerase Bst 3.0 and T7 RNA polymerase, followed by cleavage of DNA chains using DNase I. 3. The method according to p. 1, characterized in that to obtain markers of the lengths of DNA fragments in the structure of the ring matrix, a recognition site of any restrictase or nickase is set, and the corresponding restriction enzyme or nickase is added to the reaction mixture, which results in a displacement of the profile of the resulting products amplification towards shorter fragments. 4. The method according to p. 1, characterized in that to obtain markers of the lengths of DNA fragments, primers are used with a 5'-terminal sequence of any desired length and a non-complementary initial ring matrix, providing the formation of marker ladders with any step size accurate to a single nucleotide. 5. The method according to p. 1, characterized in that to obtain markers of the lengths of DNA fragments, preparations obtained by simple mixing of reaction mixtures obtained by amplification of ring matrices of different sizes are used.

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