DNA synthesis with improved yield

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), in particular 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 accomplished through the use of cell-based processes, for example by culturing bacteria in fermenters that grow the DNA to be amplified. Also, cell-free enzymatic processes for amplifying DNA from a nascent template, such as the polymerase chain reaction and strand displacement reaction, have 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. Such devices and processes are suitable for producing small amounts of DNA for testing purposes, but do not provide sufficient quantities for other purposes. Large-scale amplification and production of specific nucleic acids and proteins has mostly been performed via cell-based processes. Such methods are generally effective for producing very large volumes of product, but are expensive to set up. Furthermore, for clinical and therapeutic purposes, it is preferable to synthesize DNA in a cell-free environment.

[0004] Chemical synthesis, for example, large-scale DNA synthesis using the phosphoramidite method, is known, but is not without drawbacks. The reaction must generally be carried out in organic solvents, many of which are toxic or otherwise harmful. Another drawback of chemical synthesis is that it is not completely efficient, since after each nucleotide addition, a proportion of the growing oligonucleotide chain is capped, resulting in yield loss. Thus, the total yield loss of the nucleotide chain being synthesized increases with each nucleotide being 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 fewer nucleic acid residues, further affecting the accuracy of synthesis.

[0005] To date, biological catalysts such as polymerases have not routinely been utilized for industrial-scale production of DNA products in vitro, and reactions have been limited primarily 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, the inventors have developed a new process that involves adding new nucleotides to the reaction mixture when nucleotides are exhausted or when product concentration reaches a threshold value.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 to be amplified is required, 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 to neutralize most of their charges, thereby 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. Thus, most, but 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 clear to those skilled in the art that as soon as a nucleotide salt is dispersed in water or other solvent, the salt can dissociate in solution into anionic and cationic components.

[0009] Generally, nucleotides are provided as either lithium or sodium salts for DNA synthesis, amplification or sequencing. Lithium salts are generally preferred because they provide greater solubility and stability against repeated freezing and thawing cycles compared to sodium salts, and also provide greater reliability and extended shelf life by maintaining sterility due to the bacteriostatic activity of lithium against various microorganisms. The use of these salts is so common that a person 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 inventors have found that the species of cation present in the nucleotide salt as counterion 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 only available as lithium or sodium salts. However, as can be seen from the examples described therein, the use of alternative cations as counterions of ionic nucleotides can have a significant effect on DNA synthesis reactions. Such effects are surprising and unexpected, as they challenge the conventional understanding of the use of nucleotide salts and require the design and production of new counterion salt dNTPs to carry out the examples.

[0012] overview The present invention relates to a process for 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 would be possible under current techniques. This significantly increases productivity and at the same time reduces the cost of DNA synthesis, particularly large-scale DNA synthesis.

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

[0014] The present invention generally requires that temperature be cycled via heating and cooling during amplification. The present invention relates to an isothermal method of amplifying DNA that allows for, but does not require, the use of heat to first denature the DNA template. 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 includes the use of nucleotides in the form of salts. The salts include positive counterions (cations). The counterions are preferably monovalent cations, i.e., 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 mixtures thereof, but it is expected that at least some of the cations have an ionic radius larger than that of the sodium ion. The presence of a certain ratio of sodium or lithium in the salt or in the process is generally acceptable, but it is preferred that the salt includes 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 electrical neutrality.

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

[0017] Thus, there is provided a cell-free process for 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, the enzymatic DNA synthesis is for the production of DNA on a larger scale, i.e. for therapeutic or prophylactic use, rather than for laboratory scale amplification. This is the scale-up of the amplification on this laboratory scale, and the inventors have found that this is not as simple as providing more substrates and other components, and furthermore, that the yield is the same. In the case of nucleotide salts with sodium and lithium, they have been found to be inhibitory for DNA synthesis enzymes at higher concentrations. The inventors have found alternative ways to avoid this inhibition. The present invention allows the modification of the reaction set-up, and therefore the use of nucleotide salts at concentrations equal to or higher than 10 mM. Thus, the present process includes the use of nucleotide salts at concentrations equal to or higher than 10 mM, said concentrations being determined when the nucleotides are added. The concentrations are determined in the reaction mixture in which the present process is carried out. Thus, the concentrations of the nucleotides are determined in the reaction mixture at the time when the nucleotides are added. Thus, the concentrations are initial concentrations or concentrations at the beginning of the process.

[0019] Thus, there is provided a cell-free process for enzymatic DNA synthesis comprising the use of nucleotides provided 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, said salt comprising 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 be substituted with up to There may be four monovalent cations. Furthermore, it is possible to use mixtures of nucleotide salts 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, wherein the cell-free process is either

[0023] Nucleotides in this form may be provided at a concentration of greater than 10 mM. Thus, there is provided a cell-free process for enzymatic DNA synthesis which involves the use of nucleotides in the form of salts, said nucleotides being (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 above, wherein

[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 the enzymatic synthesis of DNA 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 above, wherein

[0026] The enzymatic DNA synthesis may include any enzyme capable of synthesizing DNA, examples of which include 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. The 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 comprising: contacting a nucleotide sequence with at least one polymerase in the presence of one or more nucleotides in a salt form to form a reaction mixture, said nucleotides being 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 above, wherein

[0029] Alternatively, the nucleotide salt is a nucleotide salt that includes, but is not limited to, a monovalent cation that is sodium or lithium, but a significant proportion of the cation has an ionic radius larger than that of the sodium ion.Therefore, 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 nucleotide is present at a concentration of at least 10 mM, and is 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). It is therefore the concentration after addition to the reaction mixture. It will be understood that 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. Furthermore, 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 substrate for enzymatic reaction.

[0031] The inventors have surprisingly found that when nucleotide salts contain monovalent cations with ionic radii larger than that of sodium ion, the need for divalent cations in synthesis is reduced. For example, during DNA synthesis reaction, for example, magnesium (divalent cation) is present in a minimum ratio of at least 1:1 with nucleotide salt. This is because magnesium is required in the active site of some polymerase enzymes, and it 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 inventors have developed a method in which the need for magnesium or other divalent cations is significantly reduced. This is important because reducing the components involved in DNA synthesis significantly reduces costs, but also, higher concentrations of magnesium are associated with reduced fidelity in DNA synthesis.

[0032] The divalent cation is Mg 2+ , B.E. 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. For 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] The present invention therefore 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 reduction in this context is compared to the same reaction in which lithium or sodium ions are present in the nucleotide salt.

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

[0035] Moreover, the method developed by the present inventors herein can be carried out under a wide range of conditions with respect to other components present. These conditions range from conventional buffering levels to effectively carrying out the reaction with the necessary components in water without providing additional buffering agents. The necessary components can include the enzymes that synthesize DNA, i.e. polymerases, nucleotide salts, and divalent cations (as salts), and optionally additional components that are required depending on the conditions of the reaction, selected from template, denaturant, pyrophosphatase, or one or more primers. These components can form a reaction mixture.

[0036] Therefore, it is advantageous to provide 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 larger than that of sodium ion, since it allows for a surprising improvement in DNA yield and / or an improvement in the efficiency of conversion of nucleotides to DNA. These improvements can be compared to similar reaction mixtures in which all nucleotides are provided as conventional salts alone, for example as lithium or sodium salts only, or as a mixture of these two ions. The provision of different nucleotide salts to those conventionally used has some further surprising advantages, such as the ability to reduce, in some cases to zero, the concentration of buffer in the reaction mixture, and to reduce the need for divalent cation cofactors, most often magnesium, for the reaction mixture.

[0037] In one form, the template directs the enzymatic DNA synthesis in the process. The template can be a DNA template. The amplification of the template is preferably via strand displacement. The amplification of the template is preferably isothermal, i.e., does not require cycling between low and high temperatures for the amplification to proceed. In this scenario, heat may be used at the beginning 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 in a range of temperatures that do not affect the denaturation of the template and the product, in order to allow any primer to enter between the double-stranded template, as the case may be. Isothermal temperature conditions require that the reaction is not heated to the point where the template and the product denature (as compared to PCR, which requires thermal cycling to denature the template and the 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 that is suitable for the enzyme.

[0038] The cell-free process preferably involves the amplification of the 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 fact that a polymerase must use two strands to extend the nascent single strand. This describes the ability of a single stranded DNA to open up 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 up the double stranded DNA and facilitates the advancement of the polymerase of the leading strand.

[0039] Independently, an optional feature of any form of the invention is that the template may be circular. Strand displacement amplification of the DNA template may be performed by rolling circle amplification (RCA). The polymerase may be Phi29, or a mutant thereof. Amplification of DNA may be isothermal, i.e., at a constant temperature. One or more primers may be random primers. Pairs or sets of primers may be used. The synthesized DNA may include concatemers that contain tandem units of the DNA sequence amplified from the DNA template. The DNA template may 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 which 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 their salts 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 their salt. Each individual nucleotide salt may contain up to four monovalent cations to maintain electrical neutrality. The nucleotide salt used in the process may contain one or more monovalent cations, i.e. one or more chemical species of monovalent cations, preferably most, if not all, of said monovalent cations have an ionic radius larger than that of 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 the nucleotide or its salt in the process, i.e. in the reaction mixture, can be preferably higher than 10 mM and up to at least 100 mM. Such concentrations are important in the production of higher yields of DNA and can be as high as 3 g / l to as high as 30 g / l, where two concentrations are indicated. Preferably, the concentrations of the nucleotide or its salt mentioned are the concentrations at the start of the process, i.e. the initial or initial concentration of the nucleotide or its salt in the reaction mixture which also contains the enzymes necessary for DNA synthesis. Subsequent addition of further components may reduce this concentration, and their use by the DNA synthesis 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 the volume of stock nucleotide salt solution / powder used when preparing the process.

[0045] The terms nucleotide and nucleotide salt are used interchangeably in the art since, by nature, all nucleotides are provided as salts. This process can be a batch process or a continuous flow process.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 of different counterions can add specific features to the enzymatic DNA synthesis reaction.For example, the use of nucleotide salts with cesium ions 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 80mM nucleotide concentration.

[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] Thus, the use of nucleotide salts containing any one of the cesium, ammonium, ammonium derivatives or rubidium cations in the enzymatic synthesis of cell-free DNA constitutes part of the present invention. Thus, the use of nucleotide salts containing these ions constitutes 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 the nucleotides, preferably at a ratio of 0.2:1 to 0.8:1 divalent cations, preferably at a ratio of 0.2:1 to 0.5:1. The ions are counterions in the nucleotide salts.

[0050] Enzymatic cell-free DNA synthesis using such ions can be performed in minimal buffers without added salts or detergents that have been shown to enhance DNA synthesis or aid primer binding. The minimal buffers may contain substances (buffers) to stabilize the pH. The minimal buffers may 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 an enzymatic method for the synthesis of nucleotides comprising the use of cesium ions. The present invention provides cell-free synthesis of DNA that is mediated by cleavage of nucleotides, where it is necessary to maintain the ratio of divalent ions to nucleotides at or below 0.5:1.

[0053] Further advantages are described below. BRIEF DESCRIPTION OF THE DRAWINGS The invention is further explained below with reference to exemplary embodiments and the accompanying drawings. [Brief description 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 MgCl2) in DNA synthesis reactions. Each plot shows the measured DNA yield obtained (g / l) versus the theoretical DNA yield (g / l) corresponding to the initial / starting total nucleotide salt concentration (mM). In all of the 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 when using cesium-dNTPs. [Diagram 2] FIG. 2 is a graph showing data from a DNA synthesis experiment, plotting the dNTP salt concentration (mM) that showed the maximum measured DNA yield against 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 the results for sections where the ratio of magnesium ion to nucleotide salt is less than 0.5, sections where the ratios are 0.5 and 1, and the final section where the ratio is greater than 1. The thresholds for these sections are also shown, with the dotted line showing a magnesium ion to nucleotide (dNTP) ratio of 0.5:1 and the solid line showing a ratio of 1:1. [FIG. 2 is a graph showing data from a DNA synthesis experiment. In the examples, DNA yields were measured for various DNA synthesis reactions carried out with a fixed starting concentration of nucleotide salt, using various counterions and increasing concentrations of magnesium chloride. The graph shows the measured DNA yield (g / l) plotted against all magnesium chloride concentrations of nucleotide salts tested.] [Diagram 3]Figure 3 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 measured DNA yield (g / l) is plotted against the initial / starting dNTP salt concentration (mM). [Figure 4] Figure 4 is a plot from a DNA synthesis experiment using rolling circle amplification of a DNA template, in which it was tested whether the presence of additional monovalent cations in the reaction mixture had an effect on the DNA synthesis reaction. In this experiment, ammonium-nucleotide salts were used, and monovalent cation chloride salts as shown were also included in the reaction mixture. The starting ratio of ammonium dNTP salt to monovalent chloride salt was 1:4, to provide one monovalent cation for each ammonium ion present on the dNTP (of which there are four). Thus, the starting ratio of ammonium ion (on the dNTP salt) to monovalent cation is 1:1. [Diagram 5] Starting concentrations of magnesium were also varied: 5 mM, 10 mM, 20 mM, and 40 mM, corresponding to 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 no added salt as a control. [Figure 6] Figures 5a and 6b are 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 (MgCl2) concentrations are 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 is a 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 ori are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 according to 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 processed to form circles, particularly minicircles, single-stranded closed circles, double-stranded closed circles, double-stranded open circles, or closed linear double-stranded DNA. The DNA may form or be processed to form specific secondary structures, such as, but not limited to, hairpin loops (stem loops), imperfect hairpin loops, pseudoknots, or any one of the 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. Using the process of the present invention, lengths of up to 77 kilobases or more are possible. 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 be more than 3g / l. The amount of DNA synthesized is preferably 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 30g / l or more. The amount of DNA synthesized is preferably 5g / l. The amount of DNA produced may be described as industrial or commercial amount in large scale or mass production. The DNA produced by the process of the present invention can be homogenous in quality, i.e., in DNA length and sequence. The process can therefore be suitable for large scale DNA synthesis. The process can be homogenous in terms of fidelity of synthesis.

[0059] Alternatively, the amount of DNA produced in a synthesis reaction may be compared to the theoretical maximum yield that would be 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 total 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 that can be obtained by the methods of the present invention are higher than 50% of the theoretical maximum, up to 90% of the theoretical maximum, and even more. Thus, the percentage of theoretical maximum yield achieved by the method of the present invention includes 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% and 95% or more. Conventionally, when using commercially available nucleotide salts, the yield achieved can be disappointing due to the effect of ions that may be inhibitory to the process.

[0060] DNA is synthesized in an enzymatic reaction. This enzymatic synthesis may involve the use of any DNA synthesis 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. This 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 mixtures thereof.

[0062] The template may comprise any sequence, either naturally occurring or artificial. The template may be of any suitable length. In particular, 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, 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 may be amplified. The template may contain sequences for expression. The DNA may be DNA for expression in cells (i.e. in vitro or in vivo transfected cells) or for expression in cell-free systems (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, said genes 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 synthesizing enzyme. 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 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, for example terminal transferases, can also be used.

[0066] Modified polymerases may also be used, which may have been 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 such 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 preferred that the polymerase has one or more characteristics suitable for manufacturing processes. 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. The polymerase preferably 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, for example, Phi29 (New England Biolabs, Inc., Ipswich, Massachusetts, USA). (New England Biolabs), Deep Vent® (New England Biolabs), Bacillus stearothermophilus (Bst) DNA polymerase Reverse transcriptase I (New England Biolabs), Klenow fragment of DNA polymerase I (New England Biolabs), M-MuLV reverse transcriptase (New England Biolabs), VentR® (exo-minus) DNA polymerase (New England Biolabs), VentR® 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 mentioned, it typically means the average number of nucleotides added by the polymerase enzyme per association / dissociation with the template, 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. Thus, the template is amplified by displacing the complementary strand and synthesizing a new complementary strand. Thus, during strand displacement replication, the newly replicated strand is displaced to force the polymerase to replicate an additional complementary strand. The amplification reaction begins when the 3' free end of the primer or 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 a primer or other strand annealed to the 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 yields of product. It is to be understood that strand displacement amplification methods differ from PCR-based methods in that double-stranded DNA is not an impediment to continued synthesis of new DNA strands, so cycles of denaturation are not essential for efficient DNA amplification. Strand displacement amplification may only require one initial round of heating if the initial template is double-stranded, if a primer is used, to denature the initial template and allow the primer to anneal to the primer binding site. Since no further heating or cooling is required after this, the amplification may be described as isothermal. In contrast, PCR methods require a denaturation cycle (i.e., increasing 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 DNA.

[0070] The strand displacement 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 displacement 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 polymerase, followed by displacing another strand, which may be the original complementary strand of the double-stranded template or a newly synthesized complementary strand, where the newly synthesized complementary strand is synthesized by the action of polymerase on the initial primer annealed to the template. Thus, the amplification of the template can be performed by displacing the replicated strand via the 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, or LAMP. LAMP typically uses 4-6 primers that recognize 6-8 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. An internal primer, containing the sequences of the sense and antisense strands of the target DNA, initiates LAMP. Subsequent strand-displacement DNA synthesis primed by the external primer releases a single-stranded DNA. This serves as a template for DNA synthesis primed by a second internal and external primer that hybridizes 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 initiates displacement DNA synthesis, resulting in 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. In the case of linear templates, the initial products of strand displacement amplification are single-stranded concatemers, which are 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 that result in the formation of concatemer-like double-stranded DNA products that contain multiple repeats of the same amplified DNA. Thus, there are multiple copies of each amplified "single unit" DNA in the concatemer-like double-stranded DNA products. RCA polymerases are particularly preferred for use in the process of the invention. The products of RCA-type strand displacement replication processes may require processing to release single units of 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), in combination with 0.2-4 mM nucleotides.

[0074] To allow amplification, according to some forms, enzymatic DNA synthesis may require one or more primers. If no template is used, the primers are designed to allow an initiation point for DNA synthesis and initiate the synthesis reaction. If a template is used, the primers may be non-specific (i.e. random in sequence) or specific to one or more sequences contained within the template. Alternatively, a primase enzyme may be provided to generate the primers de novo. If the primers are random sequence primers, they allow non-specific 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 of longer length, for example sequences of 12, 15, 18, 20 or 30 nucleotides in length. The random primers may have a length of 6-30, 8-30 or 12-30 nucleotides. Random primers are typically provided as a mix of oligonucleotides representing all possible combinations of, for example, hexamers, heptamers, octamers or nonamers on the template.

[0075] In one embodiment, the primer or one or more of the primers are specific.This means that they have a sequence that is 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 inside the two primer binding sites.Alternatively, a single specific primer can be used.A set of primers can also be adopted.

[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 may 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, contacting the template with the polymerase and one or more primers may be performed 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 includes a buffer containing 30 mM Tris-HCl pH 7.5, 20 mM KCl, 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. They 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, and thus does not require 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 as 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 include a pH buffer or, in some forms, does not include a pH buffer.

[0080] A nucleotide is a monomer, or single unit, of nucleic acid; a nucleotide is composed 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, generally, up to four are expected to be present in nucleotide salts. The monovalent cations preferably have an ionic radius larger than that of the sodium ion. The ionic radius is the radius of the ion in the ionic crystal structure. Ionic radius is typically given 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, the values ​​of ionic radius are distinct enough to provide the periodic trend recognized for atomic ions, where ionic radius increases as one moves down the group of the periodic table. For the same ion, 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. In general, ionic radius decreases with increasing positive charge. Thus, when ionic radius is mentioned herein, it may be a reference to any possible ionic radius of that ion. Exemplary ionic radii are provided in Table 6.

[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 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 + ) and especially preferred is ammonium. Ammonium has a larger ionic radius than sodium under all conditions. Also included are ammonium derivatives, and exemplary groups of these include monoalkylammonium, dialkylammonium, trialkylammonium, choline, quaternary ammonium and imidazolium. Those skilled in the art will recognize additional ammonium derivatives with a single positive charge suitable for use as counter ion 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 concentration of nucleotide salts may include any combination of the various nitrogenous bases.

[0085] Preferably, the pentose sugar is deoxyribose and therefore the nucleotides are deoxyribonucleotides. Nucleotides may be in the form of deoxynucleoside triphosphates, denoted as dNTPs. This is a preferred embodiment of the present invention. Suitable dNTPs may 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 or derivatives thereof. Preferably, a mixture of dATP, dGTP, dTTP and dCTP, or modified versions thereof, is used. Any suitable ratio of these dNTPs may 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 with a mixture of one or more suitable bases, preferably one or more of adenine (A), guanine (G), thymine (T), cytosine (C). In the 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 or 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 nucleotide-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 concentration is indicated as the concentration of nucleotide salt at the start or beginning of the process. The concentration is indicated after the addition of nucleotide / nucleotide salt, in which case the addition may be an addition to the reaction mixture. The nucleotide salt may be a mixture of any suitable nucleotide salt with various nitrogenous bases. The concentration is applied to the total amount of nucleotide salts 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 the appropriate monovalent cation as the counter ion.

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

[0090] Therefore, the nucleotide salt used in the process of the present invention may comprise a mixture of different nucleotide salts, for example a mix of potassium-nucleotide salts and cesium-nucleotide salts. A variety of different salts can be used. It is preferred that at least 75%, optionally 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the salts have a counterion with an ionic radius larger than that of sodium ion. It may be desirable to use a mixture of different salts to maximize DNA yield and to take advantage of the different characteristics of various counterions. As an alternative, 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 comprises a different monovalent cation, at least one of the cations has an ionic radius larger than that of sodium ion. Thus, two or more different nucleotide salts can be used in the process of the invention, the salts varying by the use of different counterions. All of the salts 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 the template via strand displacement is preferred. Preferably, the conditions promote amplification of the template by displacement of the replicated strand via strand displacement replication of another strand. The conditions include the use of any temperature that allows amplification of DNA, typically including temperatures in the range of 20 to 90°C. Preferred temperature ranges may be about 20 to about 40 or about 25 to about 35°C. Preferred temperatures for LAMP amplification are 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 the skilled artisan. For example, if phi29 DNA polymerase is used, the suitable temperature range is expected to be about 25 to about 35°C, preferably about 30°C. However, the thermostable phi29 may also function at higher constant temperatures. The skilled artisan can routinely ascertain the suitable temperature for efficient amplification by the process of the invention. For example, the process can be carried out at various temperatures and the yield of amplified DNA monitored to ascertain the optimal temperature range for a given polymerase. The amplification can be carried out at a constant temperature, and the process is preferably isothermal. Since strand displacement amplification is preferred, there is no need to change the temperature to separate the DNA strands. Thus, the process can be an isothermal process.

[0093] Other conditions that promote DNA synthesis would conventionally 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 in 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 buffering agents. Such buffers include, but are not limited to, MES, Bis-Tris, ADA, ACES, PIPES, MOBS, MOPS, MOPSO, Bis-Trispropane, 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] Buffers are generally defined by a mixture of reaction components. They usually include 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 detergent, e.g., Triton-X-100, to ensure optimal activity or stability of the enzyme. A minimal buffer is composed of only a buffering reagent, with no additional salt or detergent supply, although small amounts of cationic species may be present for DNA synthesis that requires 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 is one in which no pH buffer is provided or defined in the mixture of reaction components, and no additional salts or detergents are present. This "buffer-free" system contains only the reaction components necessary for DNA synthesis alone, and does not include any additional salts or detergents provided for chemical denaturation or simply nucleotide salt counterions. It contains cationic species as ions. Thus, in this system, there is no addition of additional ions 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 application of heat (exposure to 95°C for several minutes) is 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, e.g., in deionized water. Polymerase requires binding of a short oligonucleotide primer sequence to a single-stranded region of the DNA template in order 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, e.g., 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 is carried out using a divalent metal ion: 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. A maximum ratio of divalent cations to nucleotides of 2:1 is usually 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 form that is primarily used. 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. Of note, these ratios are at higher concentrations of nucleotide salts (i.e., 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, wherein the nucleotide salt comprises a counterion with an ionic radius larger than the ionic radius of the sodium ion, characterized in that 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 chain. Pyrophosphate has a similar binding affinity for magnesium ions as nucleoside triphosphates, and thus no free magnesium ions are released in the process. Using high starting concentrations of nucleotides during synthesis would result 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 the prior art.

[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, performed under conditions in which the ratio of divalent cations to dNTPs is reduced.

[0102] This effect is particularly pronounced with ammonium and cesium, or mixtures thereof, containing nucleotide salts. 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, and in particular includes bovine serum albumin (BSA) and other stabilizing proteins. The reaction conditions may also be improved by adding substances that loosen DNA and make template denaturation easier. Such substances include, for example, dimethyl sulfoxide (DMSO), formamide, glycerol, and betaine. A DNA condensing agent may also be included in the reaction mixture. Such substances include, for example, polyethylene glycol or cationic lipids or cationic polymers.

[0103] However, in certain embodiments, these components may be reduced or removed from the reaction mixture, for example, from the reaction mixture in a minimal buffer system or a 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 certain 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 MgCl2, 20 mM (NH4)2SO4, 5% glycerol, 0.2 mM BSA, 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 MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, 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 include 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 are capable of degrading pyrophosphates generated by the polymerase from dNTPs during strand replication. Accumulation of pyrophosphates during the reaction may cause inhibition of DNA polymerase and reduce the rate and efficiency of DNA amplification. Pyrophosphatases are capable of degrading pyrophosphates to non-inhibitory phosphates. An example of a pyrophosphatase suitable for use in the process of the present invention is pyrophosphatase from Saccharomyces cerevisiae, commercially available from New England Biolabs. is available at

[0108] Any single-stranded binding protein (SSBP) can be used in the process of the present invention to stabilize single-stranded DNA. SSBP is an essential component of living cells and is involved in all processes involving ssDNA, such as DNA replication, repair and recombination. In these processes, SSBP can bind to the temporarily formed ssDNA and help stabilize the ssDNA structure. An example of a SSBP suitable for use in the process of the present invention is the T4 gene 32 protein, which is 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 more than 3 g / l, more than 4 g / l, more than 5 g / l, more than 6 g / l, more than 7 g / l, more than 8 g / l, more than 9 g / l, more than 10 g / l, more than 11 g / l, more than 12 g / l, more than 13 g / l, more than 14 g / l, more than 15 g / l, more than 16 g / l, more than 17 g / l, more than 18 g / l, more than 19 g / l, more than 20 g / l, more than 21 g / l, more than 22 g / l, more than 23 g / l, more than 24 g / l, more than 25 g / l, more than 26 g / l, more than 27 g / l, more than 28 g / l, more than 29 g / l, or more 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 aim 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 allows for economical production / synthesis of DNA on an industrial scale using enzymatic processes catalyzed by DNA synthesizing enzymes or polymerases. The process of the present invention allows for efficient incorporation of nucleotides into the DNA product. It is believed that the process of the present invention allows for the scale-up of reaction mixtures from several liters to tens of liters. The improved yield, productivity or throughput can 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 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 mixtures 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, a cell-free process for synthesizing DNA includes contacting a DNA template with at least one polymerase in the presence of one or more nucleotides in a 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 larger 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 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 invention provides a cell-free process for the enzymatic synthesis of DNA, comprising the use of nucleotides provided as salts, the salts comprising monovalent cations having an ionic radius larger than that of the sodium ion, preferably the nucleotide salts are provided or present at a concentration greater than 10 mM.

[0114] The 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 the ionic radius of the potassium ion, optionally at a concentration of said 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. Working 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, ammonium, salts, stock concentration 100mM (contract synthesis) Phi29 DNA polymerase, stock concentration 2.4g / l (produced in-house) Thermostable pyrophosphatase, stock concentration 2000U / ml (Enzymatics) DNA primer, stock concentration 5 mM (Oligofactory) Plasmid template: ProTLx-K B5X4 LUX15-0-15-10-15 AT-STEM, stock concentration 0.1 g / l (produced in-house) 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). EXAMPLES

[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. It has been reported that low magnesium ion concentrations may result in little DNA synthesis, while high concentrations often cause the production of nonspecific products, as well as an increase in dNTP misincorporation and subsequent replication errors. Since magnesium binds to the phosphate moiety of each dNTP, the general 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 a promising candidate for DNA polymerase. Magnesium is an absolute requirement for high-fidelity DNA synthesis by enzymes such as ribosome inhibitors. Magnesium also binds to DNA, leading to conformational changes and the formation of cross-links between separate strands at concentrations higher than those required for DNA synthesis.

[0119] For enzymatic production of DNA in industrially relevant quantities, it is necessary to maximize the concentration of dNTPs used in the reaction to achieve maximum DNA yield. In addition, the reaction needs to be both efficient and accurate. Commercially available dNTPs are either sodium or lithium salts, typically containing four metal monovalent cations per molecule. Most publications on DNA synthesis ignore the nature of the counterions and their possible influence on the formation of magnesium-dNTPs. If the concentration of dNTPs is increased during the reaction, the concentration of monovalent counterions can increase four-fold, and therefore they have a possible effect on the DNA amplification reaction.

[0120] Therefore, understanding the dynamics of magnesium-monovalent counterions in DNA synthesis is critical to 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 Reactions were 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 48 hours after RCA, a 1.5-fold molar excess of EDTA over 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 the addition of 200 μl of 5 M NaCl. The concatemeric DNA was then precipitated by the further addition of 100 μl of 50% (w / v) PEG8000. The mixture was shaken thoroughly for 15 minutes to ensure adequate 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 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 the different dNTP salts affect the DNA yield as measured. The values ​​in brackets 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 maximum DNA yields were achieved with potassium, arsenic, and thiol, which are not commercially available. We demonstrate that this can be achieved using ammonium and cesium dNTP salts. By using the counterions of these dNTP salts 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 dNTP concentration of 20 mM. Sodium-dNTPs demonstrated superiority over their lithium equivalents, with peak DNA yields at 40 mM magnesium (6.897 g / l) achieved with 30 mM dNTPs.

[0134] Ammonium was the best dNTP counterion to achieve the highest DNA yield (13.44 g / l) at the highest starting concentration of dNTPs (50 mM dNTPs and 40 mM MgCl2) while maintaining a magnesium / dNTP ratio of 0.8. Trends in the data indicate that it is expected that it would be possible to further increase the starting concentration of ammonium-dNTPs and their incorporation into DNA by further increasing the MgCl2 concentration.

[0135] Potassium-dNTPs were also superior to their lithium and sodium counterparts in both DNA yields achieved at high dNTP concentrations (50 mM and 40 mM MgCl2) and a magnesium / dNTP ratio of 0.80. Under the reaction conditions, potassium-dNTPs performed nearly as well as ammonium-dNTPs.

[0136] At 5 mM and 10 mM MgCl2 in this process, the maximum DNA yield (5.719 g / l and 8.262 g / l) is achieved using cesium-dNTPs at starting concentrations of 25 mM and 30 mM, respectively. The magnesium / dNTP ratio at 5 mM MgCl2 and 25 mM dNTPs is 0.2, the lowest recorded value in all presented data. Thus, 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 its pK of 9.24. a It exists as 50% ammonia (NH3) in water. The volatility of NH3 allows the use of DNA processing techniques such as evaporation under low pressure that are not possible with monovalent metal cations.

[0138] FIG. 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. It is clearly shown that the dependence on magnesium is 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 concentration of magnesium required for high levels of dNTP utilization. There is a clear correlation between the number of cations and the number of cations. The 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, the selective use of dNTP salt counterions can increase DNA yields in industrial processes. This is demonstrated in Example 6 by the selective use of dNTP, DNA, and released phosphate (PO4 3- This may be due to the differential affinity of the counterions for the .DELTA.) anions and the competitive kinetics with divalent magnesium cations. EXAMPLES

[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 MgCl2.

[0145] result The results indicate that, contrary to the widely accepted view that at least a 1:1 magnesium / dNTP ratio is required for efficient dNTP incorporation, different magnesium, rather than lithium, is required. It has been shown that changing to dNTPs with counterions of different ratios can reduce magnesium levels in RCA reactions much below this ratio while still improving DNA yields.

[0146] As can be seen from 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 for 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 of lithium and sodium can reduce this ratio to as low as 0.2:1. EXAMPLES

[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, experiments were carried out in a minimal buffer consisting of 30 mM Tris-HCl (pH 7.9) alone supplemented with 5 mM MgCl2 to eliminate the effect of possible counterions of the buffer components. 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 MgCl2.

[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 the RCA proceeds even in the absence of 30 mM KCl and 5 mM (NH4)2SO4 present in the standard reaction buffer. The observed trends in yield increase with concentration of dNTP salts with various counterions are consistent with the data presented in Example 1, confirming that ammonium-dNTPs perform better than dNTP salts with other counterions.

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

[0152] [Table 8] EXAMPLES

[0153] Rolling circle amplification (RCA) reactions at different concentrations of magnesium ions and ammonium-dNTPs to determine maximum measured 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 yields by increasing concentrations of ammonium-dNTPs 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 the starting concentration of dNTPs in the reaction to be further increased (up to 80 mM) and produce extremely high levels of DNA. This is achieved by significantly increasing the concentration of MgCl2 in the minimal buffer to 80 mM. It is clear that even with 80 mM MgCl2 and 80 mM ammonium-dNTPs, peak yields of DNA were not achieved. Adding more dNTPs is expected to increase DNA yields 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. EXAMPLES

[0157] Determination of productivity limits of RCA in water-magnesium chloride mixes. Introduction and reaction setup DNA amplification experiments were then carried out at 10 mM, 20 mM and 40 mM concentrations of MgCl2, using various potassium-, cesium- and ammonium-dNTPs in reaction media that did not contain 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. Apart 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 by-product of the reaction could maintain the pH at a level that promoted 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. On the other hand, 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 unequilibrated conditions compared to the standard buffered environment (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 unequilibrated conditions. At 40 mM MgCl2 and 60 mM ammonium-dNTPs (magnesium / dNTP ratio of 0.67), High DNA yields were observed when using a 50-fold increase in the ATP / dNTP ratio. EXAMPLES

[0165] Effects 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, with MgCl2 of 5 mM, 10 mM, 20 mM, and 40 mM, respectively. To each group, LiCl, NaCl, KCl, or NH4Cl was added to a total concentration of 70 mM, 100 mM, 140 mM, and 200 mM, respectively. This caused the addition of cation concentrations in competition with the concentration of ammonium counterions of the dNTPs. In addition, the concentration of ammonium was doubled when NH4Cl was added. The magnesium / dNTP ratio for each experimental group is 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. Moreover, 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. On this basis, the presence of lithium and sodium should be avoided in industrial DNA production processes that require high concentrations of dNTPs for high DNA yields. EXAMPLES

[0170] Investigating the 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 specific 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 as 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 that the buffering capacity of ammonium dNTPs is greater than that of cesium dNTPs at dNTP salt concentrations below 30 mM, as expected. Interestingly, at concentrations of dNTP salt higher than 30 mM, the average pH of the cesium dNTP and ammonium dNTP reactions is similar at about 7 and 7.5, respectively. The data suggests that the phosphate group on the dNTP itself, when present in sufficient concentration, acts to regulate the pH to about 7. This is an advantage for industrial scale synthesis reactions that require the use of high concentrations of dNTP salts, since DNA polymerase enzymes can function efficiently at about 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.

Claims

1. 1. A cell-free process for enzymatic synthesis of DNA 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 the sodium ion; The process further comprises the use of one or more divalent metal cations. The cell-free process described above.

2. 1. A cell-free process for the 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 larger 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; Either The process further comprises the use of one or more divalent metal cations. The cell-free process described above.

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. 4. The cell-free process of any one of claims 1 to 3, wherein the one or more monovalent cations are independently selected from alkali metals, transition metals, or polyatomic ions.

5. 5. The cell-free process of claim 4, wherein the one or more monovalent cations are independently selected from the group comprising potassium, ammonium, derivatives of ammonium, rubidium, cesium, or francium.

6. The cell-free process according to any one of claims 1 to 5, further comprising the use of one or more primers or primase.

7. The cell-free process according to any one of claims 1 to 6, further comprising the use of a template.

8. 8. The cell-free process for synthesizing DNA according to any one of claims 1 to 7, wherein the one or more divalent metal cations are selected from the group comprising magnesium, manganese, calcium, beryllium, zinc and strontium.

9. 9. The cell-free process of claim 8, wherein the ratio of the divalent metal cation to the nucleotide 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 uses a maximum concentration of 10 mM of sodium and / or lithium nucleotide salts.

11. The cell-free process according to any one of claims 1 to 10, further comprising the use of a chemical denaturant.

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

14. The cell-free process according to any one of claims 1 to 13, for the synthesis of DNA on a large scale.

15. Use of nucleotide salts containing cesium cations in enzymatic cell-free DNA synthesis.

16. The use according to claim 15, wherein the enzymatic cell-free synthesis of DNA is carried out in the presence of divalent cations, at a ratio of divalent cations:nucleotides of 0.2:1 to 0.8:

1.

17. The use according to claim 15, wherein the cell-free DNA synthesis is carried out in a minimal amount of buffer.

18. 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 that contain cesium ions.

19. Use of nucleotide salts containing rubidium cations in enzymatic cell-free DNA synthesis.

20. 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, wherein the salt comprises an ammonium ion.

21. 21. The cell-free process, use, method, cell-free method or enzymatic synthesis according to any one of claims 1 to 20, wherein the enzyme is a DNA polymerase.

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