Improved yield DNA synthesis

Abstract

To provide an improved process for acellular enzymatic DNA synthesis featured by improved yield and / or improved efficiency, preferably on large scale.SOLUTION: Disclosed is a cell free process for enzymatic DNA synthesis comprising use of nucleotide salt, where the salt includes monovalent cation having an ionic radius larger than that of sodium ion. The species of cation present in the nucleotide salt as the counter-ion is critical to the yield, efficiency and fidelity of high yielding enzymatic DNA synthesis reaction.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to improved processes for the synthesis of deoxyribonucleic acid (DNA), particularly for the cell-free enzymatic synthesis of DNA with improved yields and / or improved efficiency, preferably on a large scale. [Background technology]

[0002] Amplification of deoxyribonucleic acid (DNA) can be achieved through the use of cell-based processes, such as culturing bacteria in fermentors that grow the DNA to be amplified. Cell-free enzymatic processes for amplifying DNA from a starting template, such as the polymerase chain reaction and strand displacement reaction, have also been described.

[0003] To date, DNA amplification on a test scale has been performed using devices based on microtiter plates and robotically controlled pipettes to add reaction components as needed. While these devices and processes are suitable for producing small amounts of DNA for testing purposes, they do not provide sufficient quantities for other purposes. Large-scale amplification and production of specific nucleic acids and proteins has mostly been achieved through cell-based processes. While these methods are generally effective for producing very large volumes of product, they are expensive to set up. Furthermore, for clinical and therapeutic purposes, it is preferable to synthesize DNA in a cell-free environment.

[0004] Although chemical synthesis, such as the phosphoramidite method, is known for large-scale DNA synthesis, it is not without drawbacks.This reaction generally needs to be carried out in organic solvents, many of which are toxic or otherwise harmful.Another drawback of chemical synthesis is that this synthesis is not completely efficient, because after each nucleotide addition, a proportion of the growing oligonucleotide chain is capped, resulting in yield loss.Therefore, the total yield loss of the synthesized nucleotide chain increases with each nucleotide added to the sequence.This inherent inefficiency in chemical synthesis of oligonucleotides ultimately limits the length of the oligonucleotides that can be efficiently produced to oligonucleotides with 50 or less nucleic acid residues, and further affects the accuracy of synthesis.

[0005] To date, biological catalysts such as polymerases have not traditionally been utilized for industrial-scale production of DNA products in vitro, and reactions have been primarily limited to microliter-scale volumes. Scale-up processes using enzymatic synthesis of DNA have proven problematic, particularly with disappointing yields of DNA products.

[0006] The applicant has previously addressed the ability to scale up using commercially available nucleotides. As described in WO2016 / 034849, which is incorporated herein by reference, a new process was developed that involves adding new nucleotides to the reaction mixture when nucleotides are depleted or when the product concentration reaches a threshold. However, having established that even higher yields can be achieved, the inventors have developed a new method described herein to further enhance the yield of enzymatic DNA synthesis.

[0007] Enzymatic DNA generally requires the use of a polymerase or polymerase-like enzyme to catalyze the addition of nucleotides to a nascent nucleic acid chain. A template DNA is required to amplify the DNA; however, it is also possible to perform template-free DNA synthesis, in which case incorporation occurs de novo.

[0008] It is important to note that due to the highly charged nature of nucleic acids, they are constantly surrounded by counterions, neutralizing most of their charges and reducing electrostatic repulsion between sections of the sequence so that they can be condensed into a pure, compact structure in cells. Nucleotides, the building blocks of nucleic acids, are also ionic species and require the presence of positive counterions to maintain electrical neutrality. Therefore, most, if not all, nucleotides are supplied as salts with positive counterions. Because nucleotides have four negative charges, salts are typically prepared with two divalent cations or four monovalent cations. It will be apparent to those skilled in the art that once a nucleotide salt is dispersed in water or another solvent, the salt can dissociate into anionic and cationic components in solution.

[0009] Generally, nucleotides are provided as either lithium or sodium salts for DNA synthesis, amplification, or sequencing. Lithium salts are generally preferred because they offer greater solubility and stability against repeated freezing and thawing cycles compared to sodium salts, and because lithium's bacteriostatic activity against various microorganisms provides greater reliability and extended shelf life by maintaining sterility. The use of these salts is so common that those skilled in the art would not question the presence of counterions with the nucleotides. In fact, all of the nucleotides used in the examples of WO2016 / 034849 are lithium salts of nucleotides, because they are sold as a good choice for those skilled in the art. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] WO2016 / 034849 Summary of the Invention

[0011] However, the present inventors have discovered that the cationic species present in nucleotide salts as counterions is important for the yield, efficiency, and fidelity of high-yield enzymatic DNA synthesis reactions. This is rather unexpected, since commercially available nucleotides are generally available only as lithium or sodium salts. However, as can be seen from the examples described therein, using alternative cations as counterions to ionic nucleotides can have a significant impact on DNA synthesis reactions. This effect is surprising and unexpected, as it challenges conventional understanding of the use of nucleotide salts and requires the design and production of new counterion salt dNTPs to carry out the examples.

[0012] overview The present invention relates to a process for the cell-free production or synthesis of DNA. The process allows for enhanced DNA production compared to current techniques, i.e., increased or greater yields, more efficient processes, or the ability to perform enzymatic DNA synthesis in an environment with fewer additional components than is possible under current techniques. This significantly increases productivity and simultaneously reduces the cost of DNA synthesis, particularly large-scale DNA synthesis.

[0013] Generally, the present invention relates to enzymatic DNA synthesis using polymerase enzymes or other DNA-synthesizing enzymes that may optionally be engineered to confer specific properties.

[0014] The present invention generally requires that the temperature be cycled through heating and cooling during amplification. The present invention relates to an isothermal method of amplifying DNA that allows for the use of heat to first denature the DNA template, although this does not require it. The present invention preferably relates to the use of a polymerase enzyme that is capable of replicating a DNA template via strand displacement replication, either independently or with the aid of other enzymes.

[0015] The process of the present invention involves the use of nucleotides in the form of salts. The salts contain positive counterions (cations). Preferably, these counterions are monovalent cations, i.e., they have a single positive charge due to the loss of one electron. To increase the yield and / or efficiency of DNA synthesis, the monovalent cations do not necessarily have to be sodium or lithium ions or a mixture thereof, but it is expected that at least some of the cations have an ionic radius larger than that of the sodium ion. While the presence of a certain ratio of sodium or lithium in the salt or in the present process is generally acceptable, it is preferred that the salt contains monovalent cations with an ionic radius larger than that of the sodium ion. It will be understood that nucleotides have four negative charges, and generally, four monovalent cations are present in the salt to maintain electroneutrality.

[0016] Thus, there is provided a cell-free process for the enzymatic synthesis of DNA that includes the use of nucleotides provided as salts, wherein the salts include monovalent cations with an ionic radius greater than the ionic radius of the sodium ion.

[0017] Thus, there is provided a cell-free process for the enzymatic synthesis of DNA comprising the use of nucleotides in the form of a salt, wherein the salt comprises a monovalent cation having an ionic radius greater than the ionic radius of the sodium ion.

[0018] Preferably, enzymatic DNA synthesis is for larger-scale DNA production, i.e., for therapeutic or prophylactic use, rather than experimental-scale amplification. This refers to scaling up experimental amplification, and the inventors have found that it is not as simple as providing more substrates and other components, and the yield remains the same. In the case of sodium and lithium nucleotide salts, they have been found to be inhibitory to DNA polymerases at higher concentrations. The inventors have found an alternative way to avoid this inhibition. The present invention allows for a change in the reaction setup, thus allowing the use of nucleotide salts at concentrations equal to or greater than 10 mM. Therefore, the present process involves the use of nucleotide salts at concentrations equal to or greater than 10 mM, which are determined when the nucleotides are added. The concentrations are determined in the reaction mixture in which the process is carried out. Thus, the concentrations of the nucleotides are determined in the reaction mixture at the time the nucleotides are added. Therefore, the concentrations are initial concentrations or concentrations at the start of the process.

[0019] Thus, there is provided a cell-free process for enzymatic DNA synthesis comprising the use of nucleotides supplied as salts at a concentration of at least 10 mM, wherein the salts comprise monovalent cations having an ionic radius greater than the ionic radius of the sodium ion.

[0020] Thus, there is provided a cell-free process for enzymatic DNA synthesis comprising the use of nucleotides in the form of a salt at a concentration of at least 10 mM, wherein the salt comprises a monovalent cation having an ionic radius greater than the ionic radius of the sodium ion.

[0021] Any of the nucleotide salts described herein may contain up to 100 nucleotides in order to maintain electroneutrality. There may be four monovalent cations. Furthermore, mixtures of nucleotide salts can be used in enzymatic DNA synthesis or cell-free processes.

[0022] Thus, there is provided a cell-free process for enzymatic DNA synthesis that involves the use of nucleotides provided as salts, said nucleotides comprising: (a) in the form of a salt with a single monovalent cation whose ionic radius is greater than that of the sodium ion, or (b) in the form of a salt with two or more different monovalent cations, at least one of which has an ionic radius greater than the ionic radius of the sodium ion; The present invention provides a cell-free process in which the

[0023] Nucleotides in this form may be provided at concentrations greater than 10 mM. Thus, a cell-free process for enzymatic DNA synthesis involving the use of nucleotides in the form of salts, wherein the nucleotides are (a) in the form of a salt with a single monovalent cation whose ionic radius is greater than that of the sodium ion, or (b) in the form of a salt with two or more different monovalent cations, at least one of the cations having an ionic radius greater than the ionic radius of the sodium ion; The cell-free process is provided as described above, wherein the cell-free process is any one of the following:

[0024] The nucleotide in this form is present at a concentration greater than 10 mM. "Single monovalent cation," as used herein, means a chemical species that is a single monovalent cation, up to four of which can be used to balance the negative charge in a nucleotide ion.

[0025] Alternatively, a cell-free process for enzymatic DNA synthesis comprising the use of nucleotides in the form of salts, said salts being present at a concentration of at least 10 mM; (a) in the form of a salt containing a monovalent cation whose ionic radius is greater than that of the sodium ion, or (b) two or more salt forms, each salt containing a different monovalent cation, at least one of the cations having an ionic radius greater than the ionic radius of the sodium ion; The cell-free process is provided as described above, wherein the cell-free process is any one of the following:

[0026] Enzymatic DNA synthesis may include any enzyme capable of synthesizing DNA, including polymerases or modified polymerases. The polymerase may be from any of the known DNA polymerase families, such as family A, B, C, D, X, Y, and RT. An example of a DNA polymerase from family X is terminal deoxynucleotidyl transferase.

[0027] Enzymatic DNA synthesis may be performed de novo, without the use of a template. Enzymatic DNA synthesis may involve a template, for example a DNA template.

[0028] Enzymatic DNA synthesis may be carried out in a reaction mixture containing the components described herein. Alternatively, a cell-free process for synthesizing DNA may be provided, comprising: contacting a nucleotide with at least one polymerase in the presence of one or more nucleotides in salt form to form a reaction mixture, wherein the nucleotides are present at a concentration of at least 10 mM; (a) in the form of a salt with a single monovalent cation whose ionic radius is greater than that of the sodium ion, or (b) in the form of a salt with two or more different monovalent cations, at least one of the cations having an ionic radius greater than the ionic radius of the sodium ion; The cell-free process is provided as described above, wherein the cell-free process is any one of the following:

[0029] Alternatively, the nucleotide salts include monovalent cations, including, but not limited to, sodium or lithium, with a significant proportion of cations having an ionic radius greater than that of the sodium ion. Thus, there is provided a cell-free process for synthesizing DNA, comprising contacting a DNA template with at least one polymerase in the presence of one or more nucleotides in the form of a salt with a monovalent cation to form a reaction mixture, wherein the nucleotides are present at a concentration of at least 10 mM, and are not limited to sodium or lithium.

[0030] When the concentration of nucleotide or nucleotide salt is mentioned, it is preferably the concentration of nucleotide (or its salt) at the beginning of the process, i.e., the initial or initial concentration of nucleotide (or nucleotide salt).Therefore, it is the concentration after adding to the reaction mixture.It will be understood that the addition of other components can be made during the process, and such addition may dilute the concentration of nucleotide / nucleotide salt unless additional nucleotide is provided to replenish the concentration.In addition, since nucleotide / nucleotide salt is used or consumed by the process, i.e., DNA synthesis reaction, the concentration of nucleotide / nucleotide salt is expected to decrease as the process proceeds.In certain embodiments, additional nucleotide / nucleotide salt may be added as the process proceeds to replenish substrates for enzymatic reactions.

[0031] The present inventors have surprisingly found that when a nucleotide salt contains a monovalent cation with an ionic radius larger than that of the sodium ion, the need for divalent cations in synthesis is reduced. For example, during a DNA synthesis reaction, magnesium (a divalent cation) is present at a minimum ratio of at least 1:1 with the nucleotide salt. This is because magnesium is required in the active site of some polymerase enzymes, and magnesium can form a complex with nucleotides prior to incorporation and can also form its own salt with phosphate species released during DNA synthesis. However, under certain conditions, the present inventors have developed a method that significantly reduces the need for magnesium or other divalent cations. This is important because reducing the components involved in DNA synthesis significantly reduces costs, but higher concentrations of magnesium are associated with reduced fidelity in DNA synthesis.

[0032] The divalent cation is Mg 2+ , Be 2+ , Ca 2+ , Sr 2+ , Mn 2+ or Zn 2+ and preferably Mg 2+ or Mn 2+ The ratio of metal cation to nucleotide salt may be about 1:1 in the reaction mixture. In DNA synthesis, a ratio lower than 1:1 is desirable and preferred, since a ratio higher than 1:1 may result in some imprecision in DNA synthesis. Divalent cations can be provided for enzymatic DNA synthesis in the form of any suitable salt.

[0033] Therefore, the present invention also relates to enzymatic DNA synthesis carried out under conditions of reduced divalent cations, including the use of nucleotide salts with one or more monovalent cations having an ionic radius larger than that of the sodium ion, where the reduction is compared to the same reaction in which lithium or sodium ions are present in the nucleotide salt.

[0034] The present inventors have surprisingly found that the use of nucleotide salts with alternative counterions, such as ammonium and cesium ions, in the process of the present invention reduces the need for buffers that would be incorporated into enzymatic DNA synthesis, which is advantageous because it further reduces the cost of the synthesis reaction, which may be beneficial for DNA synthesis for therapeutic use.

[0035] Furthermore, the method developed by the present inventors can be carried out under a wide range of conditions regarding the other components present. These conditions range from conventional buffering levels to those in which the reaction with the necessary components in water can be carried out effectively without the need for additional buffering. The necessary components can include an enzyme for synthesizing DNA, i.e., polymerase, nucleotide salts, and divalent cations (as salts), and optionally additional components required depending on the reaction conditions, selected from a template, a denaturant, pyrophosphatase, or one or more primers. These components can form a reaction mixture.

[0036] Therefore, providing the process, i.e., the reaction mixture, with at least a certain proportion of nucleotides as salts with monovalent positive counterions (cations) having an ionic radius greater than that of the sodium ion is advantageous because it allows for a surprising improvement in DNA yield and / or an improvement in the efficiency of nucleotide conversion to DNA. These improvements can be compared to a similar reaction mixture in which all nucleotides are provided as conventional salts alone, for example, as lithium or sodium salts alone, or as a mixture of these two ions. The provision of different nucleotide salts to those conventionally used has several further surprising advantages, such as the ability to reduce, in some cases even eliminate, the concentration of buffer in the reaction mixture, and reduce the need for divalent cation cofactors, most often magnesium, for the reaction mixture.

[0037] In one aspect, a template directs enzymatic DNA synthesis in this process. The template can be a DNA template. Template amplification is preferably via strand displacement. Template amplification is preferably isothermal, i.e., does not require cycling between low and high temperatures for amplification to proceed. In this scenario, heat may be used initially to denature the template, if necessary, or the template may be denatured by chemical means. However, once the template is denatured, the temperature may be maintained within a range that does not affect template and product denaturation, optionally allowing either primer to enter between the double-stranded template. Isothermal temperature conditions require that the reaction is not heated to the point where the template and product denature (as opposed to PCR, which requires thermal cycling to denature the template and product). Generally, such reactions are carried out at a constant temperature depending on the preference of the enzyme itself. The temperature may be any temperature suitable for the enzyme.

[0038] The cell-free process preferably involves amplification of a template via strand displacement replication. This synthesis releases single-stranded DNA, which in turn can be copied into double-stranded DNA using a polymerase. The term strand displacement refers to the need for a polymerase to double-strand the nascent single strand to extend it. This describes the ability of a single-stranded DNA to open and displace downstream DNA encountered during synthesis. DNA polymerases with varying degrees of strand displacement activity are commercially available. Alternatively, strand displacement can be achieved by providing a DNA polymerase and a separate helicase. The replicative helicase opens the double-stranded DNA, facilitating the advancement of the polymerase on the leading strand.

[0039] Independently, an optional feature of any aspect of the present invention is that the template may be circular. Strand displacement amplification of the DNA template can be performed by rolling circle amplification (RCA). The polymerase can be Phi29 or a mutant thereof. DNA amplification can be isothermal, i.e., at a constant temperature. One or more primers can be random primers. Primer pairs or sets can be used. The synthesized DNA can include concatemers containing tandem units of the DNA sequence amplified from the DNA template. The DNA template can be closed linear DNA, preferably the DNA template is incubated under denaturing conditions to form closed circular single-stranded DNA.

[0040] The amount of DNA that can be synthesized is equal to or more than 3 g per liter of reaction mixture, in particular 16 g / l or more, preferably up to 30 g / l and more.

[0041] The amount of DNA that can be synthesized can be more than 60% of the calculated maximum yield for each reaction mixture.Preferably, the amount of DNA that can be synthesized can be more than 80% of the calculated maximum yield.The calculated maximum yield is based on the theoretical yield in which all nucleotides are incorporated into the product, which can be calculated by those skilled in the art.

[0042] The efficiency of DNA synthesis from nucleotides (or nucleotide salts) may be described as the percentage of nucleotides or salts thereof supplied to the reaction mixture that are successfully incorporated into products over the course of the reaction.

[0043] The cell-free process requires at least one nucleotide. One or more additional nucleotides can then be added. The nucleotide or additional nucleotide is a deoxyribonucleoside triphosphate (dNTP), or a derivative or modified version thereof. The nucleotide or additional nucleotide is one or more of deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), and their derivatives. The nucleotide or additional nucleotide is provided as its salt. Each individual nucleotide salt may contain up to four monovalent cations to maintain electroneutrality. The nucleotide salt used in the process may contain one or more monovalent cations, i.e., one or more chemical species of monovalent cations, and it is preferred that most, if not all, of the monovalent cations have an ionic radius larger than that of the sodium ion. It will be understood that these can dissociate in solution and therefore contribute to the presence of cations in the process.

[0044] The concentration of nucleotides or their salts in the process, i.e., in the reaction mixture, is preferably higher than 10 mM and can be at least 100 mM. Such concentrations are important for the production of higher yields of DNA and can be as high as 3 g / l to 30 g / l, where two concentrations are given. Preferably, the concentrations of nucleotides or their salts mentioned are those at the start of the process, i.e., the initial or initial concentration of nucleotides or their salts in the reaction mixture, which also contains the enzymes necessary for DNA synthesis. Subsequent addition of additional components may reduce this concentration, and their use by the DNA synthesizing enzyme will also reduce the concentration from the starting concentration. Those skilled in the art will know how to calculate the concentration of nucleotides / nucleotide salts based on the other components and volume of stock nucleotide salt solution / powder used when preparing this process.

[0045] The terms nucleotide and nucleotide salt are used interchangeably in the art since, by nature, all nucleotides are supplied as salts. This process can be a batch process or a continuous flow process.The batch can be a closed batch (i.e., all reaction components are provided at the beginning of DNA synthesis), or additional components can be added to the reaction as needed during this process, for example, as described in WO2016 / 034849, which is incorporated herein by reference.If additional addition is required, this will dilute the concentration of nucleotide or nucleotide salt, unless additional nucleotide salt is added to replenish the concentration.

[0046] The inventors have found that each different counterion can add specific characteristics to the enzymatic DNA synthesis reaction.For example, the use of cesium ions in nucleotide salts allows enzymatic DNA synthesis in the presence of reduced levels of magnesium.Furthermore, the use of ammonium ions in nucleotide salts allows the use of somewhat higher concentrations of nucleotides.Examples show DNA synthesis at a nucleotide concentration of 80 mM.

[0047] The inventors have not previously recognized the use of some of these cations in nucleotide salts, as evidenced by their limited availability from commercial sources. These nucleotide salts can be custom ordered from nucleotide manufacturers, if desired.

[0048] Therefore, the use of nucleotide salts containing any one of cesium, ammonium, ammonium derivatives, or rubidium cations in enzymatic cell-free DNA synthesis is part of the present invention, and therefore the use of nucleotide salts containing these ions is part of the present invention.

[0049] Enzymatic cell-free DNA synthesis using such ions can be carried out in the presence of low levels of divalent cations, less than about 1:1, relative to nucleotides, preferably at a ratio of 0.2:1 to 0.8:1, and more preferably at a ratio of 0.2:1 to 0.5:1. The ions are counterions in nucleotide salts.

[0050] Enzymatic cell-free DNA synthesis using such ions can be performed in minimal buffers without added salts or surfactants, which have been shown to enhance DNA synthesis or aid primer binding. These minimal buffers may also contain substances (buffers) to stabilize the pH. Minimal buffers may also contain very small amounts of cations, provided by the presence of chemicals used to denature the template, such as sodium, potassium, or ammonium hydroxide. The ions are counterions in nucleotide salts.

[0051] In enzymatic DNA synthesis, low levels of magnesium ions are expected to be desirable, and the inventors have found that reliable nucleotide salts for such synthesis are nucleotide salts with cesium ions.

[0052] Thus, the present invention provides enzymatic synthesis of nucleotide salts containing cesium ions. This provides cell-free DNA synthesis, where it is necessary to maintain a divalent ion to nucleotide ratio of 0.5:1 or less.

[0053] Further advantages are described below. BRIEF DESCRIPTION OF THE DRAWINGS The invention will be further explained below with reference to exemplary embodiments and the accompanying drawings. [Brief explanation of the drawings]

[0054] [Figure 1]Figures 1A-1E are plots showing results obtained in experiments using various starting concentrations of nucleotide salts with different counterions and different initial / starting concentrations of magnesium ions (as MgCl) in DNA synthesis reactions. Each plot shows the measured DNA yield (g / L) obtained versus the theoretical DNA yield (g / L) corresponding to the initial / starting total nucleotide salt concentration (mM). In all plots, the dotted line indicates 80% efficiency of conversion of nucleotide salts to DNA, and the solid line indicates 100% efficiency of conversion. Figure 1A shows DNA synthesis results obtained using lithium-dNTPs. Figure 1B shows results achieved using sodium-dNTPs. Figure 1C shows results obtained using potassium-dNTPs. Figure 1D shows results achieved using ammonium-dNTPs. Figure 1E shows results using cesium-dNTPs. [Figure 2] Figure 2 is a graph showing data from a DNA synthesis experiment, plotting the maximum measured DNA yield as a function of dNTP salt concentration (mM) for different reaction concentrations of magnesium ion. Results are shown for nucleotide salts using lithium, sodium, potassium, ammonium, and cesium as counterions. The graph is divided into three sections, highlighting results for magnesium ion to nucleotide salt ratios less than 0.5, ratios of 0.5 and 1, and a final section where the ratio exceeds 1. The thresholds for these sections are also indicated, with the dotted line indicating a 0.5:1 magnesium ion to nucleotide (dNTP) ratio and the solid line indicating a 1:1 ratio. [Figure 3] Figure 3 is a graph showing data obtained from a DNA synthesis experiment. In the examples, the DNA yield of various DNA synthesis reactions carried out with a fixed starting concentration of nucleotide salt was measured using various counterions and increasing concentrations of magnesium chloride. The graph shows the raw DNA yield (g / L) plotted against the magnesium chloride concentration for all nucleotide salts tested. [Figure 4]Figure 4 is a graph showing data from DNA synthesis reactions using rolling circle amplification carried out in minimal buffer with various starting concentrations of nucleotide salts. The raw DNA yield (g / L) is plotted against the initial / starting dNTP salt concentration (mM). [Figure 5] Figure 5 shows plots from a DNA synthesis experiment using rolling circle amplification of a DNA template, in which the presence of additional monovalent cations in the reaction mixture was tested for their effect on the DNA synthesis reaction. In this experiment, ammonium-nucleotide salts were used, and monovalent cation chloride salts, as indicated, were also included in the reaction mixture. The starting ratio of ammonium dNTP salt to monovalent chloride salt was 1:4, providing one monovalent cation for each ammonium ion present on the dNTP (four present). Therefore, the starting ratio of ammonium ion (on the dNTP salt) to monovalent cation is 1:1. The starting magnesium concentrations were also varied: 5 mM, 10 mM, 20 mM, and 40 mM, corresponding to the ammonium counterion dNTP concentrations of 17.5 mM, 25 mM, 35 mM, and 50 mM, respectively, shown in the figure. As-measured DNA yields are plotted against the indicated concentrations of ammonium counterion dNTP (NH4-dNTP) in the presence of counterion chloride salts of monovalent cations including lithium, sodium, potassium, ammonium, and cesium, with a control containing no added salt. [Figure 6] Figures 6a and 6b show the results of several pH determination experiments comparing the pH of reaction mixtures lacking polymerase, template, and primer at various starting concentrations of nucleotide salts. Variable initial magnesium chloride (MgCl) concentrations were used. The plots show the measured pH versus the concentration of nucleotide salts at the stated initial magnesium chloride concentrations. No DNA synthesis occurred. Figure 6a shows a plot of the data for cesium-dNTPs. Figure 6b shows a plot of the data for ammonium-dNTPs. [Figure 7]7 shows the plasmid map proTLx-K B5X4 LUX ST(AT) used in the Examples. The processing site (TelRL), Luc2 reporter gene, kanamycin resistance gene, CMV promoter, and pUC oni are shown. DETAILED DESCRIPTION OF THE INVENTION

[0055] Detailed Description The present invention relates to a cell-free process for large-scale synthesis of DNA. The process of the present invention allows for high-throughput synthesis of DNA.

[0056] The deoxyribonucleic acid (DNA) synthesized by the present invention may be any DNA molecule. The DNA may be single-stranded or double-stranded. The DNA may be linear. The DNA may be engineered to form a circle, particularly a minicircle, a single-stranded closed circle, a double-stranded closed circle, a double-stranded open circle, or a closed linear double-stranded DNA. The DNA may form, or be engineered to form, specific secondary structures, such as, but not limited to, a hairpin loop (stem loop), an imperfect hairpin loop, a pseudoknot, or any one of various types of double helices (A-DNA, B-DNA, or Z-DNA). The DNA may also form hairpin and aptamer structures.

[0057] The synthesized DNA may have any suitable length. Lengths of up to 77 kilobases or more are possible using the process of the present invention. More specifically, the length of DNA that can be synthesized according to the process of the present invention may be approximately up to 60 kilobases, or up to 50 kilobases, or up to 40 kilobases, or up to 30 kilobases. Preferably, the synthesized DNA may be 100 bases to more than 77 kilobases, 500 bases to 60 kilobases, 200 bases to 20 kilobases, more preferably 200 bases to 15 kilobases, and most preferably 2 kilobases to 15 kilobases.

[0058] The amount of DNA synthesized according to the process of the present invention can exceed 3 g / L. Preferably, the amount of DNA synthesized is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 g / L or more. A preferred amount of DNA synthesized is 5 g / L. The amount of DNA produced may be described as an industrial or commercial amount for large-scale or mass production. The DNA produced by the process of the present invention can be homogeneous in quality, i.e., DNA length and sequence. This process can therefore be suitable for large-scale DNA synthesis. This process can be homogeneous in terms of synthesis fidelity.

[0059] Alternatively, the amount of DNA produced in a synthesis reaction may be compared to the theoretical maximum yield expected to be achieved if 100% of the nucleotides were incorporated into the synthesized DNA. The methods of the present invention not only improve the overall yield obtained, but also improve the efficiency of the process, meaning that more of the supplied nucleotides are incorporated into the synthesized DNA product than in previous methods. The yields obtainable by the methods of the present invention are greater than 50% of the theoretical maximum, up to 90% of the theoretical maximum, and even exceeding it. Thus, percentages of the theoretical maximum yield that can be achieved by the methods of the present invention include 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or greater. Conventionally, when using commercially available nucleotide salts, the yields achieved can be disappointing due to the effect of ions that can be inhibitory to the process.

[0060] DNA is synthesized in an enzymatic reaction. This enzymatic synthesis may involve the use of any DNA synthesizing enzyme, most often a polymerase enzyme, or a modified polymerase enzyme. These are discussed further below. DNA synthesis may be de novo synthesis, which does not require a template. Enzymatic synthesis may also require the use of a template for DNA synthesis. The template may be any suitable nucleic acid depending on the polymerase, but is preferably a DNA template.

[0061] The template can be any suitable template that simply provides instructions for DNA synthesis by containing a specific sequence. The template can be single-stranded (ss) or double-stranded (ds). The template can be linear or circular. The template can contain natural, artificial, or modified bases, or a mixture thereof.

[0062] The template may comprise any sequence, either naturally occurring or artificial. The template may have any suitable length. Specifically, the template may be up to 60 kilobases, or up to 50 kilobases, or up to 40 kilobases, or up to 30 kilobases. Preferably, the DNA template may be 10 to 100 bases, 100 to 60 kilobases, or 200 to 20 kilobases, more preferably 200 to 15 kilobases, and most preferably 2 to 15 kilobases.

[0063] The template may be provided in sufficient quantity for use in the process by any method known in the art, for example, the template may be produced by PCR.

[0064] In this process, the entire template or a selected portion can be amplified. The template may contain sequences for expression. The DNA may be DNA for expression in cells (i.e., in cells transfected in vitro or in vivo) or DNA for expression in a cell-free system (i.e., protein synthesis). The sequences for expression may be sequences for therapeutic purposes, i.e., gene therapy of DNA vaccines. The sequences for expression may be genes, which may code for DNA vaccines, therapeutic proteins, etc. These sequences may contain sequences that are transcribed into active RNA forms, i.e., short interfering RNA molecules (siRNAs).

[0065] Optionally, the template may be contacted with at least one polymerase, as described below. An enzymatic DNA synthesis reaction may require at least one DNA polymerase. Preferably, the enzyme is a polymerase. The polymerase links nucleotides together to form a DNA polymer. One, two, three, four, or five different enzymes and / or polymerases can be used. The polymerase can be any suitable polymerase from any family of polymerases that synthesizes a polymer of DNA. The polymerase can be a DNA polymerase. Any DNA polymerase can be used, including any commercially available DNA polymerase. Two, three, four, five, or more different DNA polymerases can be used, for example, one of which provides a proofreading function and one or more of which do not. DNA polymerases with different mechanisms can be used, for example, strand-displacing polymerases and DNA polymerases that replicate DNA by other methods. A suitable example of a DNA polymerase that does not have strand-displacing activity is T4 DNA polymerase. Template-independent polymerases, such as terminal transferases, can also be used.

[0066] Modified polymerases may also be used, which may be engineered to modify their characteristics, for example, to abolish their template dependence, to alter their temperature dependence, or to stabilize the enzyme for in vitro use.

[0067] The polymerase may be highly stable so that its activity is not substantially reduced by prolonged incubation under process conditions.Therefore, the enzyme preferably has a long half-life under various process conditions, including but not limited to, temperature and pH.It is also preferable that the polymerase has one or more characteristics suitable for the manufacturing process.The polymerase preferably has high fidelity, for example, by having proofreading activity.In addition, the polymerase has high processivity, high strand displacement activity, and low K for dNTPs and DNA. m Preferably, the polymerase exhibits a DNA exonuclease activity unrelated to its proofreading activity. The polymerase is capable of using circular and / or linear DNA as a template. The polymerase is capable of using dsDNA or ssDNA as a template. Preferably, the polymerase does not exhibit a DNA exonuclease activity unrelated to its proofreading activity.

[0068] One skilled in the art can determine whether a given polymerase exhibits the characteristics as defined above by comparing the characteristics exhibited by commercially available polymerases, such as Phi29 (New England Biolabs, Inc., Ipswich, MA, USA). (New England BioLabs), Deep Vent® (New England BioLabs), Bacillus stearothermophilus (Bst) DNA polymerase Examples of such polymerases include DNA polymerase I (New England Biolabs), Klenow fragment of DNA polymerase I (New England Biolabs), M-MuLV reverse transcriptase (New England Biolabs), Vent® (exo-minus) DNA polymerase (New England Biolabs), Vent® DNA polymerase (New England Biolabs), Deep Vent® (exo-) DNA polymerase (New England Biolabs), and Bst DNA polymerase large fragment (New England Biolabs). When high processivity is referred to, it typically refers to the average number of nucleotides added by a polymerase enzyme per template association / dissociation, i.e., the length of the nascent extension resulting from a single association event.

[0069] Strand displacement polymerases are preferred. Preferred strand displacement polymerases are Phi29, Deep Vent, and Bst DNA polymerase I, or any variants thereof. "Strand displacement" describes the ability of a polymerase to displace a complementary strand when it encounters a region of double-stranded DNA during synthesis. The template is thus amplified by displacing the complementary strand and synthesizing a new complementary strand. Thus, during strand displacement replication, the newly replicated strand is displaced, allowing the polymerase to replicate an additional complementary strand. The amplification reaction begins when the 3' free end of either the primer or the single-stranded template anneals to a complementary sequence on the template (both are priming events). DNA synthesis proceeds to synthesize additional If the polymerase encounters another strand annealed to the primer or template, it displaces it and continues to extend that strand. Strand displacement can release single-stranded DNA that can serve as a template for more priming events. Priming of the newly released DNA can result in hyperbranching and high product yields. It should be understood that strand displacement amplification methods differ from PCR-based methods in that double-stranded DNA does not impede continued synthesis of new DNA strands, so denaturation cycles are not essential for efficient DNA amplification. If the initial template is double-stranded, strand displacement amplification, if primers are used, may require only one initial heating round to denature the initial template and allow the primer to anneal to the primer binding site. Because no further heating or cooling is required after this, amplification is sometimes described as isothermal. In contrast, PCR methods require a denaturation cycle (i.e., raising the temperature to 94°C or higher) during the amplification process to melt the double-stranded DNA and provide new single-stranded template. During strand displacement, the polymerase displaces an already synthesized strand of DNA, and the polymerase uses the newly synthesized DNA as a template, ensuring rapid amplification of the DNA.

[0070] The strand-displacing polymerase used in the process of the invention preferably has a processivity of at least 20 kb, more preferably at least 30 kb, at least 50 kb, or at least 70 kb, or greater. In one embodiment, the strand-displacing DNA polymerase has a processivity equal to or greater than that of phi29 DNA polymerase.

[0071] Therefore, strand displacement replication is preferred. During strand displacement replication, the template is amplified by displacing the already replicated strand synthesized by the action of a polymerase, followed by displacing another strand, which can be the original complementary strand of the double-stranded template or a newly synthesized complementary strand, where this newly synthesized complementary strand is synthesized by the action of a polymerase on the initial primer annealed to the template. Thus, amplification of the template can be achieved by displacing the replicated strand via strand displacement replication of the other strand. This process may be described as strand displacement amplification or strand displacement replication.

[0072] A preferred strand-displacement replication process is loop-mediated isothermal amplification (LAMP). LAMP typically uses four to six primers that recognize six to eight distinct regions of the template DNA. Briefly, a strand-displacing DNA polymerase initiates synthesis, and two of the primers form a loop structure to facilitate subsequent rounds of amplification. LAMP is initiated by an internal primer containing the sense and antisense strand sequences of the target DNA. Subsequent strand-displacement DNA synthesis primed by the external primers releases a single-stranded DNA. This serves as a template for DNA synthesis primed by a second internal and external primer that hybridize to the other end of the target, producing a stem-loop DNA structure. In subsequent LAMP cycles, one internal primer hybridizes to the loop on the product and primes displacement DNA synthesis, generating the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. Modified LAMP procedures may be employed, in which fewer internal primers are required.

[0073] A preferred strand displacement replication process is rolling circle amplification (RCA). The term RCA describes the ability of an RCA-type polymerase to proceed continuously around a circular DNA template strand while extending a hybridized primer. This results in the formation of a linear single-stranded product with multiple repeats of the amplified DNA. The sequence of the circular template (single unit) is repeated multiple times within the linear product. For linear templates, the initial products of strand displacement amplification are single-stranded concatemers, which can be either sense or antisense, depending on the polarity of the template. These linear single-stranded products serve as the basis for multiple hybridization, primer extension, and strand displacement events, resulting in the formation of concatemer-like double-stranded DNA products containing multiple repeats of the amplified DNA. Thus, multiple copies of each amplified "single-unit" DNA are present in the concatemer-like double-stranded DNA products. RCA polymerases are particularly preferred for use in the processes of the present invention. Products of RCA-type strand displacement replication processes may require processing to release single-unit DNA. This is desirable if single units of DNA are required. Typical strand displacement conditions using Phi29 DNA polymerase include high levels of magnesium ions, e.g., 10 mM magnesium (usually as the chloride salt), combined with 0.2-4 mM nucleotides.

[0074] In some embodiments, enzymatic DNA synthesis may require one or more primers to allow amplification. When no template is used, primers are designed to allow a starting point for DNA synthesis and initiate the synthesis reaction. When a template is used, primers may be nonspecific (i.e., random in sequence) or specific to one or more sequences contained within the template. Alternatively, a primase enzyme may be supplied to generate primers de novo. When primers are random-sequence primers, they allow nonspecific initiation at any site on the template. This allows for highly efficient amplification through multiple initiation reactions from each template strand. Examples of random primers are hexamers, heptamers, octamers, nonamers, decamers, or sequences longer in length, such as 12, 15, 18, 20, or 30 nucleotides in length. Random primers may be 6-30, 8-30, or 12-30 nucleotides in length. Random primers are typically provided as a mix of oligonucleotides representing all possible combinations of, for example, hexamers, heptamers, octamers or nonamers in the template.

[0075] In one embodiment, one or more primers are specific.This means that they have a sequence complementary to the sequence in the template that is required to initiate amplification.In this embodiment, a pair of primers can be used to specifically amplify the part of the DNA template that is inside the two primer binding sites.Alternatively, a single specific primer can be used.A set of primers can also be used.

[0076] The primer may be any nucleic acid composition. The primer may be unlabeled or may contain one or more labels, such as radionuclides or fluorescent dyes. The primer may also contain chemically modified nucleotides. For example, the primer may be capped, i.e., by chemical or physical means, to prevent the initiation of DNA synthesis until the cap is removed. The length / sequence of the primer can typically be selected based on temperature considerations, i.e., to be able to bind to the template at the temperature used in the amplification process.

[0077] In certain embodiments, the contact of the template with the polymerase and one or more primers may be carried out under conditions that promote annealing of the primer to the template. The conditions include the presence of single-stranded nucleic acid that allows hybridization of the primer. The conditions also typically include temperature and buffer that allow annealing of the primer to the template. Suitable annealing / hybridization conditions The conditions can be selected depending on the nature of the primer. An example of a conventional annealing condition that can be used in the present invention is a buffer containing 30 mM Tris-HCl pH 7.5, 20 mM KCl, and 8 mM MgCl2. However, the inventors have described herein conditions in which the buffer and divalent metal ion components are reduced but still allow primer binding. These are discussed further below. Annealing can be performed using heat after denaturation, followed by stepwise cooling to the desired reaction temperature.

[0078] However, amplification using strand displacement replication can also be performed without primers, thus eliminating the need for hybridization and primer extension. Instead, the single-stranded template self-primes by forming a hairpin with a free 3' end available for extension. The remaining steps of the amplification remain the same.

[0079] The template and / or polymerase also contact with nucleotides in the form of nucleotide salts. Combining the DNA template, polymerase, and nucleotide salts may be described as forming a reaction mixture. The reaction mixture may also include one or more primers or primases. The reaction mixture may also independently include one or more divalent metal cations. The reaction mixture may further include a chemical denaturant. Such a denaturant may be potassium hydroxide, ammonium hydroxide, or sodium hydroxide. The reaction mixture may further include an additional enzyme, such as a helicase or pyrophosphatase. The reaction mixture may contain a pH buffer, or in some embodiments, does not contain a pH buffer.

[0080] A nucleotide is a monomer, or single unit, of nucleic acid; a nucleotide consists of a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group. Any suitable nucleotide can be used.

[0081] Nucleotides exist as salts with monovalent cations. Monovalent cations are ionic species with a single positive charge, and therefore, up to four are generally expected to be present in a nucleotide salt. Preferably, the monovalent cation has an ionic radius greater than that of the sodium ion. The ionic radius is the radius of the ion in the ionic crystal structure. Ionic radius is typically expressed in units of either picometers (pm) or angstroms (Å). Ionic radius is not a fixed property of a given ion, but varies depending on various parameters such as coordination number and spin state. Nevertheless, ionic radius values ​​are distinct enough to provide the periodic trend recognized for atomic ions, where ionic radius increases down the group of the periodic table. For the same ion, the ionic radius increases with increasing coordination number, and ions in low spin states are expected to be smaller than the same ion in high spin states. Generally, ionic radius decreases with increasing positive charge. Therefore, when ionic radius is referred to herein, it can refer to all possible ionic radii of that ion. Table 6 provides exemplary ionic radii.

[0082] Nucleotides are salts of monovalent metal ions, such as, but not limited to, alkali metals (Group 1): lithium (Li + ), sodium (Na + ), potassium (K + ), rubidium (Rb + ), Cesium (Cs + ) or francium (Fr + Alternatively, or in addition, the monovalent metal ion may include a transition metal (Group 11): copper (Cu + ), silver (Ag + ), gold (Au + ) or Roentgenium (Rg + Alkali metals are preferred, and thus preferred counterions may be potassium (K+), rubidium (Rb+), cesium (Cs+), or francium (Fr+).

[0083] Nucleotides may also include salts of polyatomic monovalent ions. A polyatomic ion is an ion that contains more than one atom. This distinguishes a polyatomic ion from a monoatomic ion, which contains only one atom. Exemplary monovalent polyatomic cations include ammonium (NH4 + ) and hydronium (HO + ) are included, with ammonium being particularly preferred. Ammonium has a larger ionic radius than sodium under all conditions. Also included are ammonium derivatives, exemplary groups of which include monoalkylammonium, dialkylammonium, trialkylammonium, choline, quaternary ammonium, and imidazolium. Those skilled in the art will recognize additional ammonium derivatives with a single positive charge that are suitable for use as counterions in nucleotide salts.

[0084] The nitrogenous bases may be adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). The nitrogenous bases may also be modified bases, such as 5-methylcytosine (m5C), pseudouridine (Ψ), dihydrouridine (D), inosine (I), and 7-methylguanosine (m7G). The nitrogenous bases may also be artificial bases. The nucleotide salt concentration may include any combination of various nitrogenous bases.

[0085] Preferably, the five-carbon sugar is deoxyribose and therefore the nucleotide is a deoxyribonucleotide. Nucleotides may be in the form of deoxynucleoside triphosphates, designated dNTPs. This is a preferred embodiment of the present invention. Suitable dNTPs include dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dTTP (deoxythymidine triphosphate), dUTP (deoxyuridine triphosphate), dCTP (deoxycytidine triphosphate), dITP (deoxyinosine triphosphate), dXTP (deoxyxanthosine triphosphate), and derivatives and modified versions thereof. Preferably, dNTPs include one or more of dATP, dGTP, dTTP, or dCTP, or modified versions thereof. Preferably, a mixture of dATP, dGTP, dTTP, and dCTP, or modified versions thereof, is used. Any suitable ratio of these dNTPs can be used according to the needs of the reaction.

[0086] The nucleotides or nucleotide salts may be in the form of a solution or may need to be supplied as a solid, for example, as a powder. The nucleotides or nucleotide salts may include modified nucleotides. The nucleotides or nucleotide salts may be provided as a mixture of one or more suitable bases, preferably one or more of adenine (A), guanine (G), thymine (T), and cytosine (C). In this process, two, three, or preferably all four nucleotides (A, G, T, and C) are used to synthesize DNA. All of these nucleotides or nucleotide salts may be present in substantially equal amounts, or one, two, or more of them may be provided depending on the nature of the DNA to be synthesized.

[0087] The nucleotides may be all natural nucleotides (i.e., unmodified), they may be modified nucleotides that act like natural nucleotides and are biologically active (i.e., LNA nucleotides - locked nucleic acids), they may be modified and biologically inactive, or they may be a mixture of unmodified and modified nucleotides and / or a mixture of biologically active and biologically inactive nucleotides. Each type of nucleotide (i.e., base) may be provided in one or more forms, That is, they may be provided in unmodified and modified, or biologically active and biologically inactive forms. All of these nucleotides are capable of forming suitable salts.

[0088] In one embodiment of the present invention, the nucleotide or nucleotide salt is present at a concentration of at least 10 mM. According to this embodiment, the nucleotide or nucleotide salt may be present in the reaction mixture at a concentration of more than 10 mM, more than 15 mM, more than 20 mM, more than 25 mM, more than 30 mM, more than 35 mM, more than 40 mM, more than 45 mM, more than 50 mM, more than 55 mM, more than 60 mM, more than 65 mM, more than 70 mM, more than 75 mM, more than 80 mM, more than 85 mM, more than 90 mM, more than 95 mM, or more than 100 mM. Such concentrations are indicated as the concentration of nucleotide salt at the start or beginning of the process. The concentrations are indicated after the addition of nucleotide / nucleotide salt, in which case the addition may be to the reaction mixture. The nucleotide salt may be a mixture of any suitable nucleotide salt with various nitrogenous bases. The concentrations apply to the total amount of nucleotide salt present at the beginning of the process, regardless of their composition. Thus, for example, a nucleotide salt at a concentration of 10 mM can be any mixture of dCTP, dATP, dGTP and dTTP with appropriate monovalent cations as counter ions.

[0089] It will be understood that nucleotides supplied as salts can dissociate in water or other solvents to form anionic nucleotide entities and cations. It is a preferred part of all aspects of the present invention that the nucleotide salts are formed with counterions having an ionic radius larger than that of the sodium ion. However, polymerases or DNA synthesizers are likely to tolerate some concentration of lithium and / or sodium nucleotide salts. Therefore, there may be a portion of the nucleotide salts included in the process of the present invention in which the counterion is sodium and / or lithium. This portion is preferably less than 25%, and optionally 20%, 15%, 10%, 5%, 1% or less. Polymerases or DNA synthesizers may also tolerate sodium and / or lithium from other sources, such as denaturants. The total concentration of lithium ions in the reaction mixture does not exceed 15 mM, preferably 10 mM, and even more preferably 5 mM, 4 mM, 3 mM, 2 mM, 1 mM or less. Because lithium is considered more inhibitory, it is preferred that this ion be substantially excluded from the reaction mixture. In the case of sodium ions, the presence of sodium ions is acceptable because sodium hydroxide is commonly used as a denaturant.

[0090] Therefore, the nucleotide salts used in the process of the present invention may comprise a mixture of different nucleotide salts, for example, a mixture of potassium-nucleotide salts and cesium-nucleotide salts. A variety of different salts can be used. Preferably, at least 75%, optionally 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the salts have counterions with an ionic radius greater than that of the sodium ion. It may be desirable to use a mixture of different salts to maximize DNA yield and take advantage of the different characteristics of various counterions. Alternatively, there is provided a cell-free process for synthesizing DNA, comprising: contacting a DNA template with at least one polymerase in the presence of one or more nucleotides in salt form to form a reaction mixture, wherein the nucleotides are in the form of two or more salts, each salt comprising a different monovalent cation, at least one of the cations having an ionic radius greater than that of the sodium ion. Thus, two or more different nucleotide salts can be used in the process of the present invention, the salts varying by the use of different counterions, all of which have an ionic radius larger than that of the sodium ion. It may be preferable to require the use of a counterion with a radius.

[0091] Enzymatic DNA synthesis can be maintained under conditions that promote DNA synthesis, which will depend on the particular method selected. Amplification of a template via strand displacement is preferred. Preferably, the conditions promote amplification of the template by displacement of a replicated strand via strand displacement replication of another strand. The conditions include the use of any temperature that allows DNA amplification, generally ranging from 20 to 90°C. Preferred temperature ranges can be from about 20 to about 40°C or from about 25 to about 35°C. A preferred temperature for LAMP amplification is from about 50 to about 70°C.

[0092] Typically, the appropriate temperature for enzymatic DNA synthesis is selected based on the temperature at which a specific polymerase has optimal activity. This information is generally available and forms part of the general knowledge of those skilled in the art. For example, when phi29 DNA polymerase is used, the preferred temperature range is expected to be about 25 to about 35°C, preferably about 30°C. However, thermostable phi29 may function at higher, constant temperatures. Those skilled in the art can routinely determine the appropriate temperature for efficient amplification by the process of the present invention. For example, the process can be performed at various temperatures and the yield of amplified DNA monitored to determine the optimal temperature range for a given polymerase. Amplification can be performed at a constant temperature; the process is preferably isothermal. Because strand displacement amplification is preferred, no temperature changes are necessary to separate the DNA strands. Therefore, the process can be an isothermal process.

[0093] Other conditions that promote DNA synthesis are conventionally considered to include the presence of a suitable buffer / pH and other factors necessary for enzyme performance or stability. Suitable conventional conditions include any conditions used to provide activity of polymerase enzymes known in the art.

[0094] For example, the pH of the reaction mixture may be within the range of 3 to 10, preferably 5 to 8, or about 7, e.g., about 7.5. The pH may be maintained within this range by the use of one or more buffers, including, but not limited to, MES, Bis-Tris, ADA, ACES, PIPES, MOBS, MOPS, MOPSO, Bis-Tris propane, BES, TES, HEPES, DIPSO, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS, phosphate, citrate-sodium hydrogen phosphate, citrate-sodium citrate, sodium acetate-acetic acid, imidazole, and sodium carbonate-sodium bicarbonate.

[0095] A buffer is generally defined by a mixture of reaction components. It typically includes a buffering agent to maintain a stable pH; one or more additional salts composed of cationic and anionic species, i.e., sodium chloride, potassium chloride; and / or a surfactant, e.g., Triton-X-100, to ensure optimal enzyme activity or stability. A minimal buffer consists of only buffering reagents, with no additional salts or surfactants, although small amounts of cationic species may be present for DNA synthesis requiring chemical denaturation. Surprisingly, the use of higher concentrations of nucleotide salts in the process of the present invention allows the use of these minimal buffers.

[0096] A "buffer-free" system does not provide or define a pH buffer in the mixture of reaction components, and does not have any added salts or detergents. This "buffer-free" system contains only the reaction components necessary for DNA synthesis alone, and does not contain any nucleotide salts or surfactants provided for chemical denaturation or simply as a counterbalance. It contains cationic species as ions. Therefore, in this system, no additional ions are added other than those that serve a specific purpose in the DNA synthesis reaction. The counterions provided with the nucleotides (as salts) serve to stabilize the nucleotides prior to use in the process.

[0097] Although the application of heat (exposure to 95°C for several minutes) has been used to denature double-stranded DNA, other approaches more suitable for DNA synthesis can be used. Double-stranded DNA can be easily denatured by exposure to high or low pH environments, or when cations are absent or present in very low concentrations, such as in deionized water. Polymerase requires the binding of a short oligonucleotide primer sequence to a single-stranded region of the DNA template to initiate its replication. The stability of this interaction, and therefore the efficiency of DNA synthesis, depends in particular on the presence of metal cations, particularly divalent cations, such as Mg, which can be considered an essential part of the process.2+ It may be affected by the concentration of ions.

[0098] Enzymatic DNA synthesis may also require divalent metal ions. This process involves the use of divalent metal ions, such as magnesium (Mg 2+ ), manganese (Mn 2+ ), calcium (Ca 2+ ), Beryllium (Be 2+ ), zinc (Zn 2+ ) and strontium (Sr 2+ The divalent ions most often used in DNA synthesis are magnesium or manganese.

[0099] Enzymatic DNA synthesis can be performed at lower concentrations of divalent metal ions than previously thought possible. Usually, a maximum ratio of divalent cations to nucleotides of 2:1 is considered necessary or optimal, and as the data in the examples show, this is particularly true for nucleotide salts with lithium ions, which are the most commonly used form. However, when alternative ions are used for these salts, the need for divalent ions, particularly magnesium, is dramatically reduced, so that the ratio of ions to nucleotide salts is approximately 1.5:1, or about 1:1, or even lower. Results are obtained when the ratio of magnesium to nucleotide salts is 0.2:1, and these results are obtained using nucleotide salts with cesium. Notably, these ratios are at higher concentrations of nucleotide salts (i.e., concentrations of 20 mM or more). Therefore, the present invention is also directed to the synthesis of DNA in which the ratio of magnesium ions to nucleotide salt is 1:1 or less, the nucleotide salt comprises a counterion with an ionic radius larger than that of the sodium ion, and the concentration of the nucleotide salt is greater than 25 mM, greater than 30 mM, greater than 35 mM, greater than 40 mM, greater than 45 mM, greater than 50 mM, greater than 55 mM, greater than 60 mM, greater than 65 mM, greater than 70 mM, greater than 75 mM, greater than 80 mM, greater than 85 mM, greater than 90 mM, greater than 95 mM, or greater than 100 mM.

[0100] During synthesis, polymerase releases pyrophosphate from nucleotides, which is incorporated into the growing DNA strand. Pyrophosphate has a similar binding affinity for magnesium ions as nucleoside triphosphates, so free magnesium ions are not released during this process. Using high starting concentrations of nucleotides during synthesis results in reduced levels of free magnesium ions. Because these ions may be required for polymerase catalytic activity, conventional thinking is that suboptimal levels resulting from interactions with phosphate or phosphate groups are likely to be detrimental to efficient amplification. Sufficient, and therefore excessive, concentrations of magnesium ions were thought to be important for DNA yield and amplification. Therefore, the ability to reduce magnesium levels while maintaining yield is an attractive improvement over conventional techniques.

[0101] Thus, the present invention provides enzymatic DNA synthesis involving the use of nucleotide salts with one or more monovalent cations having an ionic radius larger than that of the sodium ion, carried out under conditions in which the ratio of divalent cations to dNTPs is reduced.

[0102] This effect is particularly pronounced when using nucleotide salts containing ammonium and cesium, or mixtures thereof. In certain embodiments, a detergent may be included in the reaction mixture. Examples of suitable detergents include Triton X-100™, Tween 20™, and any derivatives thereof. A stabilizer may also be included in the reaction mixture. Any suitable stabilizer may be used, particularly bovine serum albumin (BSA) and other stabilizing proteins. Reaction conditions can also be improved by adding a substance that loosens DNA and makes template denaturation easier. Examples of such substances include dimethyl sulfoxide (DMSO), formamide, glycerol, and betaine. A DNA condensing agent may also be included in the reaction mixture. Examples of such substances include polyethylene glycol, cationic lipids, or cationic polymers.

[0103] However, in certain embodiments, these components may be reduced or eliminated from the reaction mixture, for example, in a minimally buffered or buffer-free system.

[0104] It is understood that those skilled in the art can modify and optimize the synthesis conditions for the process of the present invention using these additional components and conditions based on their general knowledge. Similarly, the specific concentrations of particular substances can be selected based on previous examples in the art and can be further optimized based on general knowledge.

[0105] As an example, a suitable reaction buffer used in RCA-based methods in the art is 50 mM Tris-HCl, pH 7.5, 10 mM MgCl, 20 mM (NH)SO, 5% glycerol, 0.2 mM BSA, and 1 mM dNTPs. A preferred reaction buffer used in the RCA amplification of the present invention is 30 mM Tris-HCl pH 7.9, 30 mM KCl, 7.5 mM MgCl, 10 mM (NH)SO, 4 mM DTT, and 2 mM dNTPs. This buffer is particularly suitable for use with Phi29 DNA polymerase.

[0106] A suitable reaction buffer for use with the nucleotide salts of the invention is 30 mM Tris-HCl, pH 7.9, 5 mM (NH4)2SO4, and 30 mM KCl. Under certain circumstances, enzymatic DNA synthesis may be carried out in water ("buffer-free").

[0107] Enzymatic DNA synthesis may also involve the use of one or more additional proteins. The DNA template may be amplified in the presence of at least one pyrophosphatase, such as yeast inorganic pyrophosphatase. Two, three, four, five, or more different pyrophosphatases may be used. These enzymes can degrade pyrophosphates generated by polymerase from dNTPs during strand replication. Accumulation of pyrophosphate during the reaction can cause inhibition of DNA polymerase and reduce the rate and efficiency of DNA amplification. Pyrophosphatases can degrade pyrophosphates to non-inhibitory phosphates. An example of a pyrophosphatase suitable for use in the process of the present invention is Saccharomyces cerevisiae pyrophosphatase, commercially available from New England Biolabs. is available at

[0108] Any single-strand binding protein (SSBP) can be used to stabilize single-stranded DNA in the process of the present invention. SSBPs are essential components of living cells and are involved in all processes involving ssDNA, such as DNA replication, repair, and recombination. In these processes, SSBPs can bind to transiently formed ssDNA and help stabilize the ssDNA structure. An example of an SSBP suitable for use in the process of the present invention is the T4 gene 32 protein, commercially available from New England Biolabs.

[0109] The yield of the reaction is related to the amount of DNA synthesized. The expected yield from the process according to the invention can be greater than 3 g / l. The amount of DNA synthesized is preferably or may be greater than 3 g / L, greater than 4 g / L, greater than 5 g / L, greater than 6 g / L, greater than 7 g / L, greater than 8 g / L, greater than 9 g / L, greater than 10 g / L, greater than 11 g / L, greater than 12 g / L, greater than 13 g / L, greater than 14 g / L, greater than 15 g / L, greater than 16 g / L, greater than 17 g / L, greater than 18 g / L, greater than 19 g / L, greater than 20 g / L, greater than 21 g / L, greater than 22 g / L, greater than 23 g / L, greater than 24 g / L, greater than 25 g / L, greater than 26 g / L, greater than 27 g / L, greater than 28 g / L, greater than 29 g / L, or greater than 30 g / L. The preferred amount of synthesized DNA is 5 g / L. The present invention improves the yield possible from enzymatic synthesis of DNA. The objective of the present invention is to improve the yield of cell-free enzymatic DNA synthesis processes so that DNA can be synthesized on a large scale in a cost-effective manner. The present invention makes it possible to economically produce / synthesize DNA on an industrial scale using an enzymatic process catalyzed by a DNA polymerase or polymerase. The process of the present invention allows for the efficient incorporation of nucleotides into the DNA product. It is believed that the process of the present invention allows for the reaction mixture to be scaled up to several liters to tens of liters. The improved yield, productivity, or throughput may be similar to the same reaction mixture in which all of the nucleotides are supplied as conventional salts (sodium and / or lithium).

[0110] In one embodiment, the present invention relates to a process for enhancing the synthesis of DNA, which can be equivalent to an identical reaction mixture except that all of the nucleotide salts used are exclusively sodium or lithium, or a mixture thereof.

[0111] In one aspect, the invention provides a cell-free process for synthesizing DNA, comprising contacting a DNA template with at least one polymerase in the presence of one or more nucleotides in the form of a salt with one or more monovalent cations to form a reaction mixture, wherein the nucleotides are present at a concentration of at least 10 mM, and the cations are not limited to sodium or lithium.

[0112] Alternatively, there is provided a cell-free process for synthesizing DNA, comprising contacting a DNA template with at least one polymerase in the presence of one or more nucleotides in salt form to form a reaction mixture, wherein the nucleotides are present at a concentration of at least 10 mM; (a) in the form of a salt with a single monovalent cation whose ionic radius is greater than that of the sodium ion, or (b) in the form of a salt with two or more different monovalent cations wherein at least one of the cations has an ionic radius greater than the ionic radius of a sodium ion.

[0113] The concentrations of nucleotides referred to herein are the starting concentrations of nucleotides at the start of the process, preferably the initial concentrations when the reaction mixture is formed. The present invention may also relate to a cell-free process for synthesizing DNA, comprising contacting a DNA template with at least one polymerase in the presence of one or more nucleotides in the form of a salt with sodium ion at a concentration of 10-20 mM, or up to 30 mM. The present invention also provides a cell-free process for enzymatic DNA synthesis that comprises the use of nucleotides provided as salts, wherein the salts comprise monovalent cations with an ionic radius larger than that of the sodium ion, and preferably wherein the nucleotide salts are provided or present at a concentration greater than 10 mM.

[0114] The present invention further provides enzymatic DNA synthesis carried out under conditions of reduced divalent cations, preferably magnesium, including the use of nucleotide salts with one or more monovalent cations having an ionic radius larger than that of the sodium ion.

[0115] The invention may alternatively be practiced using a nucleotide salt with a counterion having an ionic radius greater than that of the potassium ion, optionally at a concentration of the nucleotide salt greater than 25 mM, greater than 30 mM, greater than 35 mM, greater than 40 mM, greater than 45 mM, greater than 50 mM, greater than 55 mM, greater than 60 mM, greater than 65 mM, greater than 70 mM, greater than 75 mM, greater than 80 mM, greater than 85 mM, greater than 90 mM, greater than 95 mM, or greater than 100 mM.

[0116] Nucleotides in the form of salts are also referred to herein as nucleotide salts. The invention will now be described with reference to some non-limiting examples. Example material and method reagent In the examples presented, the following reagents were used: dNTP salts, lithium salt, stock concentration 100 mM (Bioline) dNTP salts: sodium, potassium, cesium, and ammonium salts, stock concentration 100 mM (contract synthesis) Phi29 DNA polymerase, stock concentration 2.4 g / l (in-house production) Thermostable pyrophosphatase, stock concentration 2000 U / ml (Enzymatics) DNA primers, stock concentration 5 mM (Oligofactory) Plasmid template: ProTLx-K B5X4 LUX15-0-15-10-15 AT-STEM, stock concentration 0.1 g / l (in-house production) Nuclease-free water (Sigma Aldrich) Magnesium chloride, stock concentration 2M (Sigma-Aldrich) Tris-Base (Thermo Fisher Scientific) Tris-HCl (Sigma-Aldrich) NaCl (Sigma-Aldrich) EDTA, stock concentration 0.5M (Sigma-Aldrich) PEG8000 (Applichem) Ethanol (Thermo Fisher Scientific) GeneRuler 1 kb+ DNA Ladder (Thermo Fisher Scientific) TAE buffer from 20x stock (Thermo Fisher Scientific) Potassium chloride (Sigma-Aldrich) Lithium chloride (Sigma-Aldrich) Cesium chloride (Sigma-Aldrich) Ammonium chloride (Sigma-Aldrich) Ammonium sulfate (Thermo Fisher Scientific). [Example]

[0117] Rolling circle amplification (RCA) reactions at different concentrations of magnesium ions and nucleotide salts (dNTP salts) (with lithium, sodium, potassium, cesium, and ammonium cations as counter ions); effect on DNA yield.

[0118] introduction The concentration of magnesium ions in the reaction buffer is important for optimal DNA synthesis by DNA polymerase. Low magnesium ion concentrations may result in little DNA synthesis, while high concentrations have been reported to often cause the production of nonspecific products, as well as an increase in dNTP misincorporation and subsequent replication errors. Because magnesium binds to the phosphate moiety of each dNTP, common practice is to use a magnesium ion concentration equal to or higher than the concentration of dNTPs used (Dean, FB, Nelson, JR, Giesler, TL, and Lasken, RS (2001). Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification. Genome Research, 11(6), 1095-1099. http: / / doi.org / 10.1101 / gr.180501). Magnesium-dNTPs are essential for DNA polymerization. Magnesium is an absolute requirement for high-fidelity DNA synthesis by enzymes. Magnesium also binds to DNA, causing conformational changes and allowing cross-links to form between separate strands at concentrations higher than those required for DNA synthesis.

[0119] For enzymatic production of DNA in industrially viable quantities, it is necessary to maximize the concentration of dNTPs used in the reaction to achieve maximum DNA yield. Additionally, the reaction must be both efficient and accurate. Commercially available dNTPs are typically either sodium or lithium salts containing four monovalent metal cations per molecule. Most publications on DNA synthesis ignore the nature of the counterion and its potential effect on the formation of magnesium-dNTPs. When the concentration of dNTPs is increased during the reaction, the concentration of monovalent counterions can increase fourfold, thus potentially affecting the DNA amplification reaction.

[0120] Therefore, understanding the dynamics of magnesium-monovalent counterions in DNA synthesis is important for maximizing DNA yields at the lowest possible magnesium concentrations to allow for maximum fidelity of the DNA product.

[0121] The following set of experiments evaluated the effect of increasing initial magnesium concentrations (5 mM, 10 mM, 20 mM, and 40 mM) and different salts of dNTPs on the yield of DNA amplified by RCA (rolling circle amplification).

[0122] Reaction setup The reaction was set up at a 100 μl scale as follows: the denaturing mix was prepared and left at room temperature while the reaction mix was assembled. These were then mixed, and DNA polymerase and pyrophosphatase were added. Table 1 shows the experimental protocol.

[0123] RCA reactions were incubated at 30°C for a minimum of 48 hours before processing.

[0124] [Table 1]

[0125] Sample processing procedure Forty-eight hours after RCA, a 1.5-fold molar excess of EDTA relative to MgCl2 was added, and the reaction was brought to a volume of 800 μl with water. They were shaken thoroughly for 15 minutes and placed on a rotator until the reaction was well mixed. The reaction was then brought to 1 ml in 1 M NaCl by adding 200 μl of 5 M NaCl. The concatemeric DNA was then precipitated by adding an additional 100 μl of 50% (w / v) PEG 8000. The mixture was shaken thoroughly for 15 minutes to ensure sufficient precipitation, and then spun at 13,000 rpm for 10 minutes in a benchtop centrifuge. The supernatant was carefully decanted, and the pellet was washed with 500 μl of 100% ethanol. The pellet was centrifuged again at 13,000 rpm for 10 minutes in a benchtop centrifuge, and the ethanol supernatant was carefully decanted. The pellet was allowed to dry for 5 minutes to evaporate residual ethanol, resuspended in 1 ml of water and placed on a rotator overnight.

[0126] Reaction DNA concentrations were quantified from UV absorption measurements using an Implen NP80 nanophotometer. Data were corrected for a 10-fold increase in reaction volume, and concentrations are expressed as g / l of dNTPs used (g) relative to the original volume (l).

[0127] result Tables 2-5 and Figures 1 and 2 show that the initial concentration of magnesium and the starting concentrations of different dNTP salts affect the measured DNA yield. The values ​​in parentheses refer to the magnesium / dNTP ratio at the maximum DNA yield achieved for each type of dNTP salt.

[0128] [Table 2]

[0129] [Table 3]

[0130] [Table 4]

[0131] [Table 5]

[0132] The data in Tables 2-5 show that the maximum yield of DNA is achieved with potassium, ammonium, and ammonium hydroxide, which are not commercially available. We demonstrate that this can be achieved using ammonium and cesium dNTP salts. By using these dNTP salt counterions and increasing the magnesium concentration to 40 mM, it is possible to use starting concentrations of dNTPs up to 50 mM and achieve efficient conversion to DNA by Phi29 DNA polymerase.

[0133] Lithium-dNTPs were poor substrates for DNA synthesis, requiring higher levels of magnesium than other monovalent cations. In fact, peak DNA yields at 40 mM magnesium (4.328 g / L) occurred only at a 20 mM dNTP concentration. Sodium-dNTPs demonstrated superiority over lithium equivalents, with peak DNA yields at 40 mM magnesium (6.897 g / L) achieved using 30 mM dNTPs.

[0134] Ammonium was the best dNTP counterion to achieve the highest DNA yield (13.44 g / L) at the highest starting dNTP concentration (50 mM dNTP and 40 mM MgCl) while maintaining a magnesium / dNTP ratio of 0.8. Trends in the data indicate that it is expected that further increases in the MgCl concentration will further increase the starting concentration of ammonium-dNTPs and their incorporation into DNA.

[0135] Potassium-dNTPs also outperformed their lithium and sodium counterparts in both DNA yields achieved at high dNTP concentrations (50 mM and 40 mM MgCl) and a magnesium / dNTP ratio of 0.80. Under the reaction conditions, potassium-dNTPs performed nearly as well as ammonium-dNTPs.

[0136] In this process, the highest DNA yields (5.719 g / L and 8.262 g / L) are achieved using cesium-dNTPs at starting concentrations of 25 mM and 30 mM, respectively, at 5 mM MgCl and 10 mM MgCl. The magnesium / dNTP ratio at 5 mM MgCl and 25 mM dNTP is 0.2, the lowest recorded value in all data presented. Therefore, the use of cesium-dNTPs is advantageous (for the outcome of the DNA amplification process) under conditions where it is beneficial to use the lowest possible concentration of magnesium ions, while still producing high yields.

[0137] The ammonium ion is unique among the other monovalent cations investigated in that it is polyatomic and completely nonmetallic. It can act as a pH buffer, with a pK of 9.24. a It exists as 50% ammonia (NH3) in water. The volatility of NH3 allows for the use of DNA processing techniques such as evaporation under low pressure, which is not possible with metal monovalent cations.

[0138] Figure 1 is a graphical representation of the data shown in Tables 2-5, showing plots of the resulting measured DNA yield (g / l) versus the theoretical DNA yield (g / l) corresponding to the initial / starting total nucleotide salt concentration (mM) at different concentrations of magnesium chloride.

[0139] Figure 2 is a plot of dNTP salt concentration (mM) showing maximum measured DNA yields for different reaction concentrations of magnesium ions. The dependence on magnesium is clearly shown to be greatest for lithium-dNTPs and sodium-dNTPs, but is reduced for other counterions.

[0140] Table 6 contains the ionic radii of monovalent counterions at different coordination numbers. The relationship between counterion size (relative to magnesium) and the magnesium concentration required for high levels of dNTP utilization. There is a clear correlation between the two. Larger cations such as potassium, cesium and ammonium are far superior to sodium and especially lithium.

[0141] [Table 6]

[0142] References: http: / / abulafia.mt.ic.ac.uk / shannon / ptable.php , Shriver and Atkins . Therefore, selective use of dNTP salt counterions can increase DNA yield in industrial processes. This is demonstrated in Example 6 by comparing the dNTP, DNA, and released phosphate (PO 3- ) anions and the competitive dynamics with divalent cations of magnesium. [Example]

[0143] Rolling circle amplification (RCA) reactions at different concentrations of magnesium ions and fixed concentrations of dNTP salts (with lithium, sodium, potassium, cesium, and ammonium cations as counterions); effect on DNA yield.

[0144] Introduction and reaction setup This experiment was designed to determine the minimum concentration of magnesium ions required for incorporation of a fixed amount of dNTPs (10 mM) in the process at the start. RCA reactions and treatments were carried out as described in Example 1. The concentration of dNTPs (as lithium, sodium, potassium, cesium, and ammonium salts) was fixed at 10 mM in the process at the start, and reactions were carried out in the standard RCA buffer used in Example 1 supplemented with 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM MgCl.

[0145] result The results suggest that, contrary to the widely accepted view that a magnesium / dNTP ratio of at least 1:1 is required for efficient dNTP incorporation, different magnesium, rather than lithium, is required. It has been shown that changing to dNTPs with counterions containing 2-methyl-3-methyl-2-pyridine can reduce the magnesium level in the RCA reaction much below this ratio, while still improving DNA yield.

[0146] As can be seen in Figure 3, the yields for both sodium-dNTPs and lithium-dNTPs are strongly dependent on magnesium levels. While there is a slight reduction in DNA produced at 2 mM MgCl2 with potassium-dNTPs, this dNTP salt form, along with cesium-dNTPs and ammonium-dNTPs, shows a lower dependence on magnesium ion concentration. This suggests that the common assumption that the optimal magnesium / dNTP ratio is 1:1 is a misconception that does not take into account the type of dNTP counterion. The data show that the use of alternative counterions, lithium and sodium, can reduce this ratio to as low as 0.2:1. [Example]

[0147] Rolling circle amplification (RCA) reactions in minimal buffer with fixed levels of magnesium ions and increasing concentrations of dNTP salts (with lithium, sodium, potassium, and ammonium cations as counterions); effect on DNA yield.

[0148] Introduction and reaction setup Next, to eliminate the effect of possible counterions of the buffer components, experiments were performed in a minimal buffer consisting of 30 mM Tris-HCl (pH 7.9) alone supplemented with 5 mM MgCl. These reactions tested the effect of increasing starting dNTP salt concentrations (2.5 mM to 20 mM dNTPs supplied as lithium, sodium, potassium, or ammonium salts) in reactions containing 5 mM MgCl.

[0149] [Table 7]

[0150] DNA processing and quantification was carried out as described in Example 1. The results are shown in Figure 4. The data show that RCA proceeds even in the absence of 30 mM KCl and 5 mM (NH)SO present in the standard reaction buffer. The observed trend in increasing yield with concentration of dNTP salts with various counterions is consistent with the data presented in Example 1 and confirms that ammonium-dNTPs perform better than dNTP salts with other counterions.

[0151] FIG. 4 demonstrates that varying the counterion of the dNTP salt allows the RCA to proceed at higher concentrations of dNTPs, with a concomitant increase in yield.

[0152] [Table 8] [Example]

[0153] Rolling circle amplification (RCA) reactions at different concentrations of magnesium ions and ammonium-dNTPs to determine the maximum intact DNA yield Introduction and reaction setup These reactions were aimed at extending the experimental data presented in Example 3 (Figure 4) and finding limits on DNA yield by increasing the concentration of ammonium-dNTP in the presence of different magnesium concentrations. RCA reactions and DNA treatments were carried out in minimal buffers essentially as described in Example 3.

[0154] result

[0155] [Table 9]

[0156] The data demonstrate that using dNTPs with ammonium counterions allows for further increasing the starting concentration of dNTPs in the reaction (up to 80 mM) and producing extremely high levels of DNA. This is achieved by significantly increasing the MgCl2 concentration in the minimal buffer to 80 mM. It is clear that peak DNA yields were not achieved even with 80 mM MgCl2 and 80 mM ammonium-dNTPs. Adding more dNTPs is expected to increase DNA yield even further. Increasing the concentrations of MgCl2 and ammonium-dNTPs (beyond 80 mM) is expected to result in even higher levels of DNA under conditions where the magnesium / dNTP ratio is predicted to be <1. [Example]

[0157] Determination of productivity limits of RCA in water-magnesium chloride mixes. Introduction and reaction setup DNA amplification experiments were then carried out with various potassium-, cesium-, and ammonium-dNTPs in reaction media containing 10 mM, 20 mM, and 40 mM MgCl2, but not Tris buffer or other salts conventionally required for optimal DNA amplification. In this respect, lithium and sodium-dNTPs were omitted because both outperformed other cations during selection. Aside from magnesium and dNTP counterions, the only other cations in the reaction included 5 mM sodium ions from the NaOH used for template denaturation. Experiments were performed to determine whether the dNTPs themselves and the phosphate byproducts of the reaction could maintain the pH at a level that promotes Phi29 DNA polymerase activity and maintain the physicochemical conditions necessary for effective DNA priming.

[0158] result

[0159] [Table 10]

[0160] DNA processing and quantification was carried out essentially as described in Example 1.

[0161] [Table 11]

[0162] [Table 12]

[0163] [Table 13]

[0164] The experimental data show that reactions performed with potassium-dNTPs produced variable results in the absence of Tris buffer. Meanwhile, both cesium-dNTPs and ammonium-dNTPs produced progressively higher DNA yields with increasing concentrations of magnesium ions and dNTPs. Cesium-dNTPs performed significantly better under these un-equilibrated conditions compared to standard buffered environments (see Table 5). High DNA yields were recorded with 40 mM MgCl2 and 50 mM cesium-dNTPs (magnesium / dNTP ratio of 0.80). There was no significant difference between DNA yields using ammonium-dNTPs in buffered or un-equilibrated conditions. At 40 mM MgCl2, 60 mM ammonium-dNTPs (magnesium / dNTP ratio of 0.67) yields were significantly higher. High DNA yields were observed when using a 2000 ng / dNTP ratio. [Example]

[0165] Effect of other counterion salts on DNA amplification using ammonium-dNTPs Introduction and reaction setup This experiment was performed to demonstrate the effect of lithium, sodium, and potassium cations on the yield of DNA obtained by RCA using ammonium-dNTPs.

[0166] [Table 14]

[0167] Reactions were set up as shown in Table 14. Four experimental groups were run containing starting concentrations of ammonium-dNTPs of 17.5 mM, 25 mM, 35 mM, and 50 mM, along with 5 mM, 10 mM, 20 mM, and 40 mM MgCl, respectively. To each group, LiCl, NaCl, KCl, or NH4Cl was added to total concentrations of 70 mM, 100 mM, 140 mM, and 200 mM, respectively. This caused the addition of cation concentrations to compete with the concentration of ammonium counterions of the dNTPs. Furthermore, when NH4Cl was added, the concentration of ammonium was doubled. The magnesium / dNTP ratio in each experimental group was less than 1.0.

[0168] DNA processing and quantification was carried out essentially as described in Example 1. The results are shown in Figure 5. Figure 5 shows that cesium, ammonium, and potassium ions are not inhibitory to DNA synthesis when ammonium-dNTPs are used. Furthermore, the ammonium concentration can still be doubled without affecting DNA yield.

[0169] Conversely, lithium and sodium are inhibitory, with lithium being more inhibitory than sodium. Based on this, their presence should be avoided in industrial DNA production processes that require high concentrations of dNTPs for high DNA yields. [Example]

[0170] Examination of buffering of DNA synthesis reactions with dNTPs Introduction and reaction setup This experiment was performed to examine the ability of dNTP salts to buffer the reaction mixture in the absence of any particular buffering agent.

[0171] [Table 15]

[0172] [Table 16]

[0173] [Table 17]

[0174] The reaction components were mixed in the ratios shown in the table above to give a final volume of 50 μl, and the pH of the mixture was then measured using a Mettler Toledo SevenCompact™ S220 pH meter equipped with an InLab® micro pH electrode.

[0175] Figure 6 shows the measured pH for various dNTP concentrations (cesium and ammonium salts) in the presence of 10 mM, 20 mM, and 40 mM MgCl2. The NaOH concentration was used to denature the template DNA used in the DNA synthesis reaction. For the purposes of this experiment, all other DNA synthesis reaction components were omitted because they were known not to affect the starting pH. In all cases, no specific pH-stabilizing buffer (e.g., Tris) was added. It is clear, as expected, that the buffering capacity of ammonium dNTPs is greater than that of cesium dNTPs at dNTP salt concentrations below 30 mM. Interestingly, at dNTP salt concentrations higher than 30 mM, the average pH of the cesium dNTP and ammonium dNTP reactions is similar, approximately 7 and 7.5, respectively. The data suggest that the phosphate group on the dNTP itself, when present in sufficient concentration, acts to regulate the pH to approximately 7. This is an advantage for industrial-scale synthesis reactions, which require the use of high concentrations of dNTP salts, because DNA polymerase enzymes can function efficiently at approximately pH 7. Importantly, this indicates that for industrial-scale reactions, DNA synthesis can be carried out without specific buffers or at low concentrations to achieve high productivity. Scope of claims at the time of international application [Section 1] 1. A cell-free process for enzymatic DNA synthesis comprising the use of nucleotide salts, wherein the salts comprise monovalent cations having an ionic radius greater than the ionic radius of the sodium ion. [Section 2] 2. The cell-free process of claim 1, wherein the nucleotide salt is present at a concentration greater than 10 mM. [Section 3] 1. A cell-free process for enzymatic DNA synthesis comprising the use of nucleotide salts, said nucleotide salts being present at a concentration of at least 10 mM; (a) a nucleotide salt containing a monovalent cation whose ionic radius is greater than that of the sodium ion, or (b) two or more nucleotide salts, each salt containing a different monovalent cation, at least one of the cations having an ionic radius greater than the ionic radius of the sodium ion; The cell-free process is any one of the above. [Section 4] 4. The cell-free process of claim 1, wherein the nucleotide salt is present at a concentration of at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM or at least 40 mM. [Section 5] 5. The cell-free process of claim 1, wherein the one or more monovalent cations are independently selected from alkaline earth metals, transition metals, or polyatomic ions. [Section 6] 6. The cell-free process of claim 5, wherein the one or more monovalent cations are independently selected from the group comprising potassium, ammonium, derivatives of ammonium, rubidium, cesium, or francium. [Section 7] 7. The cell-free process according to any one of claims 1 to 6, further comprising the use of one or more primers or primases. [Section 8] 8. The cell-free process according to claim 1, further comprising the use of a template. [Section 9] 9. The cell-free process for synthesizing DNA according to any one of claims 1 to 8, further comprising the use of one or more divalent metal cations, preferably selected from the group comprising magnesium, manganese, calcium, beryllium, zinc, and strontium. [Section 10] 10. The cell-free process of claim 9, wherein the ratio of divalent metal cations to nucleotides in the reaction mixture is 1:1 or less than 1:1, preferably less than 1:1. [Section 11] 11. The cell-free process of claim 1, wherein the process uses a maximum concentration of sodium and / or lithium nucleotide salts of 10 mM. [Section 12] 12. The cell-free process according to any one of claims 1 to 11, further comprising the use of a chemical denaturant, preferably sodium hydroxide, potassium hydroxide or ammonium hydroxide, and pyrophosphatase. [Section 13] 13. The cell-free process of claim 12, wherein no pH buffers are added, and preferably no additional salts or detergents are added. [Section 14] 14. The cell-free process of claim 13, wherein the nucleotide salt comprises a cesium ion. [Section 15] 13. The cell-free process of claim 12, wherein a pH buffer is added but no additional salts or surfactants are added. [Section 16] 16. The cell-free process of claim 15, wherein the nucleotide salt comprises an ammonium ion. [Section 17] 17. A cell-free process according to any one of claims 1 to 16 for the synthesis of DNA on a large scale, preferably at least 3 g / l. [Section 18] Use of nucleotide salts containing cesium cations in enzymatic cell-free DNA synthesis. [Section 19] 19. The use of claim 18, wherein the enzymatic cell-free DNA synthesis is carried out in the presence of low levels of divalent cations, optionally at a divalent cation:nucleotide ratio of 0.2:1 to 0.8:1, preferably 0.2:1 to 0.5:1. [Section 20] 19. The use of claim 18, wherein the cell-free DNA synthesis is carried out in a minimal amount of buffer, optionally comprising a pH buffer alone, without surfactants or added salts. [Section 21] 1. A method for amplifying a DNA template using a DNA polymerase, which requires maintaining a ratio of divalent cations to nucleotides in the reaction mixture of 0.5:1 or less, and which involves the use of nucleotide salts containing cesium ions. [Section 22] Use of nucleotide salts containing rubidium cations in enzymatic cell-free DNA synthesis. [Section 23] 1. A method for amplifying a DNA template in a cell-free environment, comprising contacting the template and a DNA polymerase with a nucleotide in the form of a salt in an amount equal to or greater than 40 mM, preferably greater than 60 mM, or optionally greater than 80 mM, wherein the salt comprises an ammonium ion. [Section 24] Enzymatic DNA synthesis carried out under conditions of reduced concentrations of divalent cations, preferably magnesium, comprising the use of nucleotides in the form of salts containing monovalent cations having an ionic radius larger than that of the sodium ion. [Section 25] 25. The cell-free process, use, method, cell-free method or enzymatic synthesis according to any one of claims 1 to 24, wherein the enzyme is a DNA polymerase, optionally a strand-displacing polymerase.

Claims

1. 1. A cell-free process for enzymatic DNA synthesis comprising the use of a nucleotide salt present at a concentration greater than 10 mM, said salt comprising a monovalent cation having an ionic radius greater than the ionic radius of a sodium ion, said monovalent cation being selected from the group comprising alkali metals or ammonium; The cell-free process further includes the use of divalent magnesium cations and the enzyme is a DNA polymerase. The above cell-free process.

2. 1. A cell-free process for enzymatic synthesis of DNA comprising the use of nucleotide salts, said nucleotide salts being present at a concentration of at least 10 mM; (a) a nucleotide salt containing a monovalent cation having an ionic radius greater than that of the sodium ion; or (b) two or more nucleotide salts, each salt containing a different monovalent cation, at least one of the cations having an ionic radius greater than the ionic radius of the sodium ion; wherein the monovalent cation is selected from the group comprising alkali metals or ammonium, the cell-free process further comprises the use of divalent magnesium cations, and the enzyme is a DNA polymerase. The above cell-free process.

3. 3. The cell-free process of claim 1 or 2, wherein the nucleotide salt is present at a concentration of at least 15 mM.

4. A cell-free process described in any one of claims 1 to 3, wherein the nucleotide salt is present at a concentration of at least 20 mM.

5. The cell-free process of claim 1, wherein the nucleotide salt is present at a concentration of at least 30 mM.

6. 6. The cell-free process of any one of claims 1 to 5, wherein the one or more monovalent cations are independently selected from the group comprising potassium, ammonium, or cesium.

7. The cell-free process according to any one of claims 1 to 6, further comprising the use of one or more primers or primases.

8. A cell-free process according to any one of claims 1 to 7, wherein enzymatic DNA synthesis occurs in a reaction mixture.

9. 9. The cell-free process of claim 8, wherein the ratio of divalent magnesium cations to nucleotides in the reaction mixture is 1:1 or less than 1:

1.

10. 10. The cell-free process of any one of claims 1 to 9, wherein the process further uses a maximum concentration of 10 mM of sodium and / or lithium nucleotide salts.

11. The cell-free process of any one of claims 1 to 10, further comprising the use of chemical denaturants and pyrophosphatase.

12. No pH buffer is added, 12. The cell-free process of claim 11, wherein the nucleotide salt comprises a cesium ion.

13. A pH buffer is added, but no additional salts or surfactants are added; 12. The cell-free process of claim 11, wherein the nucleotide salt comprises an ammonium ion.

14. A cell-free process according to any one of claims 1 to 13 for the synthesis of DNA on a large scale at at least 3 g / l.

15. The cell-free process of claim 1, wherein the DNA polymerase is a strand-displacing polymerase.

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