Synthesis of DNA at improved yield

Abstract

The present invention relates to an improved method for the synthesis of deoxyribonucleic acids (DNAs), in particular the cell-free enzymatic synthesis of DNA, preferably on a large scale, with increased yield and / or increased efficiency. Cationic species present as counterions in nucleotide salts are crucial for yield, efficiency, and accuracy of high yield enzymatic DNA synthesis reactions. The methods herein use alternative cations as counterions for ionic nucleotides, allowing for the use of higher concentrations of nucleotides in DNA synthesis, and further allowing for the use of more advantageous reaction conditions.

Description

[0001] This application is a divisional application of the application filed on August 16, 2019, with application number 2019800678114, entitled "Synthesizing DNA with Increased Yield". Technical Field

[0002] This invention relates to an improved method for synthesizing deoxyribonucleic acid (DNA), particularly cell-free enzymatic synthesis of DNA, preferably on a large scale, which has increased yield and / or increased efficiency. Background Technology

[0003] Deoxyribonucleic acid (DNA) amplification can be performed using cell-based methods, such as culturing bacteria that propagate the DNA to be amplified in a fermenter. Cell-free enzymatic methods for amplifying DNA from a starting template, including polymerase chain reaction (PCR) and strand displacement reaction (SCR), have also been described.

[0004] In the past, assay-scale DNA amplification has been performed using devices based on microtiter plates and automated pipettes to add the required reaction components. Such devices and methods are suitable for producing small amounts of DNA molecules for assay purposes, but cannot provide sufficient quantities for other purposes. Large-scale amplification and production of specific nucleic acids and proteins are mostly carried out using cell-based methods. Such methods are generally efficient for the production of very large quantities of products, but the manufacturing costs are high. Furthermore, for clinical and therapeutic purposes, DNA synthesis in a cell-free environment is preferred.

[0005] Large-scale DNA synthesis using chemical synthesis methods, such as phosphoramide synthesis, is known, but it is not without its drawbacks. The reaction must typically be carried out in organic solvents, many of which are toxic or otherwise harmful. Another disadvantage of chemical synthesis is that it is not entirely efficient, as a certain percentage of the growing oligonucleotide chain is capped with each addition of nucleotides, resulting in yield loss. Therefore, the total yield loss of synthesized nucleotide chains increases with each nucleotide added to the sequence. This inherent inefficiency of chemically synthesized oligonucleotides ultimately limits the length of oligonucleotides that can be efficiently produced to those with 50 or fewer nucleic acid residues, further impacting the accuracy of the synthesis.

[0006] To date, biocatalysts such as polymerases have not been routinely developed for the industrial-scale production of DNA products in vitro, and the reactions are largely limited to microliter-scale volumes. Scale-up methods for using enzymatic DNA synthesis have proven problematic, particularly in terms of disappointing DNA product yields.

[0007] The applicant has previously solved the ability to amplify using commercially available nucleotides. A novel method was developed, as described in WO2016 / 034849, which involves adding fresh nucleotides to the reaction mixture when fresh nucleotides are depleted or the product concentration reaches a threshold; this document is incorporated herein by reference. However, it has been determined that even higher yields can be achieved, and the inventors have developed a novel method described herein to further increase the yield of enzymatic DNA synthesis.

[0008] Enzymatic DNA synthesis typically requires the use of polymerases or polymerase-like enzymes to catalyze the addition of nucleotides to the nascent nucleic acid chain. Usually, a template DNA needs to be amplified in the reaction. However, template-free DNA synthesis is also possible, where integration occurs de novo.

[0009] It is important to note that due to the highly charged nature of nucleic acids, they are often surrounded by counterions to neutralize most of their charge, reducing electrostatic repulsion between parts of the sequence and thus enabling them to condense into neat, compact structures within the cell. The building blocks of nucleic acids (nucleotides) are also ionic and require the presence of positive counterions to maintain electroneutrality. Therefore, most (if not all) nucleotides are provided as salts with positive counterions. Since nucleotides have four negative charges, salts are typically prepared with two divalent cations or four monovalent cations. It will be apparent to those skilled in the art that once a nucleotide salt is dispersed in water or another solvent, the salt can dissociate in solution into anionic and cationic components.

[0010] Nucleotides are typically provided as lithium or sodium salts for DNA synthesis, amplification, or sequencing. Lithium is generally preferred because these salts have greater solubility and stability to repeated freeze-thaw cycles than sodium salts, and remain sterile due to lithium's antimicrobial activity against a wide range of microorganisms, thus providing greater reliability and a longer shelf life. The use of these salts is so common that those skilled in the art would have no doubt about the counterions present alongside nucleotides. In fact, all the nucleotides used in the examples of WO2016 / 034849 are lithium salts of nucleotides, as these have been commercially available as the best choice for those skilled in the art.

[0011] However, the inventors have discovered that the cationic substances present in nucleotide salts as counterions are crucial for the yield, efficiency, and accuracy of high-yield enzymatic DNA synthesis reactions. This is quite unexpected, as commercially available nucleotides are typically only available in lithium or sodium salt form. However, as can be seen from the examples included herein, using alternative cations as counterions to ionic nucleotides can have a significant impact on DNA synthesis reactions. This effect is surprising and unexpected because it challenges the conventional understanding of the use of nucleotide salts and necessitates the design and production of novel counterion salts (dNTPs) for the purposes of these examples. Summary of the Invention

[0012] This invention relates to a method for cell-free production or synthesis of DNA. Compared to current methods, this method enables increased DNA production, i.e., increased or higher yields than currently considered, a more efficient method, or the ability to perform enzymatic DNA synthesis in an environment with fewer additional components. This significantly improves productivity while reducing the cost of DNA synthesis, particularly in large-scale production.

[0013] Generally, the present invention relates to enzymatic DNA synthesis using polymerases or other DNA synthases, any of which may optionally be engineered to impart specific properties to them.

[0014] This invention generally relates to an isothermal method for amplifying DNA that does not require temperature cycling through heating and cooling during amplification, but instead allows the use of heat to initially denature the DNA template. Preferably, this invention relates to the use of polymerases capable of replicating the DNA template independently or with the aid of other enzymes via strand displacement replication.

[0015] The method of this invention relates to the use of nucleotides in the form of salts. The salt comprises a positive counterion (cation). Preferably, the counterion is a monovalent cation, i.e., it has a single positive charge due to the loss of an electron. To increase the yield and / or efficiency of DNA synthesis, the monovalent cation may not be limited to sodium or lithium ions or mixtures thereof, but at least a portion of the cations has an ionic radius larger than that of a sodium ion. The presence of a certain proportion of sodium or lithium in the salt or in the method is generally tolerable, but it is preferred that the salt contains monovalent cations with ionic radii larger than that of sodium ions. It should be understood that since nucleotides have four negative charges, typically four monovalent cations will be present in the salt to maintain electroneutrality.

[0016] Therefore, a cell-free method for enzymatic synthesis of DNA is provided, which includes using a nucleotide provided as a salt, wherein the salt comprises a monovalent cation with an ionic radius greater than that of a sodium ion.

[0017] Therefore, a cell-free method for enzymatic synthesis of DNA is provided, which includes using nucleotides in the form of salts, wherein the salts contain monovalent cations with ionic radii greater than that of sodium ions.

[0018] Preferably, enzymatic DNA synthesis is used for the large-scale production of DNA, i.e., for therapeutic or preventative purposes, rather than for laboratory-scale amplification. In such scale-up laboratory amplification, the inventors found that it is not as simple as providing more substrate and other components, and the yield is also affected. Nucleotide salts containing sodium and lithium were found to inhibit DNA synthase at higher concentrations. The inventors discovered alternative approaches to this inhibition. The present invention allows for changes in the reaction setup and therefore allows the use of nucleotide salts at concentrations equal to or greater than 10 mM. Thus, the method involves using nucleotide salts at a concentration equal to or greater than 10 mM, which is determined upon addition of nucleotides. The concentration in the reaction mixture used to carry out the method is determined. Therefore, the concentration of nucleotides in the reaction mixture is determined upon addition of nucleotides. Thus, the concentration is either the initial concentration or the concentration at the start of the method.

[0019] Therefore, a cell-free method for enzymatic DNA synthesis is provided, comprising using a nucleotide provided as a salt at a concentration of at least 10 mM, wherein the salt comprises a monovalent cation with an ionic radius greater than that of a sodium ion.

[0020] Therefore, a cell-free method for enzymatic DNA synthesis is provided, comprising using nucleotides in the form of salts at a concentration of at least 10 mM, wherein the salts comprise monovalent cations with an ionic radius greater than that of sodium ions.

[0021] As described herein, any nucleotide salt can contain up to four monovalent cations to maintain electroneutrality.

[0022] Furthermore, mixtures of nucleotide salts can be used in enzymatic DNA synthesis or cell-free methods.

[0023] Therefore, a cell-free method for enzymatic DNA synthesis is provided, the method comprising using a nucleotide provided as a salt, wherein the nucleotide is: (a) In the form of a salt having a single monovalent cation, the ionic radius of which is greater than that of a sodium ion, or (b) A salt having two or more different monovalent cations, wherein at least one cation has an ionic radius greater than that of a sodium ion.

[0024] The nucleotides in this aspect can be provided at a concentration greater than 10 mM.

[0025] Therefore, a cell-free method for enzymatic DNA synthesis is provided, which involves using nucleotides in salt form, wherein the nucleotides are: (a) In the form of a salt having a single monovalent cation, the ionic radius of which is greater than that of a sodium ion, or (b) A salt having two or more different monovalent cations, wherein at least one cation has an ionic radius greater than that of a sodium ion.

[0026] In this aspect, nucleotides are present at a concentration greater than 10 mM.

[0027] As used in this article, "single monovalent cation" refers to a single monovalent cation species, of which up to four can be used to counteract the negative charge on the nucleotide ion.

[0028] Alternatively, provide the following: A cell-free method for enzymatic synthesis of DNA, the method comprising using nucleotides in salt form, wherein the salt is present at a concentration of at least 10 mM and is: (a) A salt containing a monovalent cation whose ionic radius is greater than that of a sodium ion, or (b) Two or more salts, each containing a different monovalent cation, wherein at least one cation has an ionic radius greater than that of a sodium ion.

[0029] Enzymatic DNA synthesis can involve any enzyme capable of synthesizing DNA, including polymerases or modified polymerases. Polymerases can come from any known family of DNA polymerases, including families A, B, C, D, X, Y, and RT. An example of a DNA polymerase from family X is a terminal deoxynucleotidyl transferase.

[0030] Enzymatic DNA synthesis can be carried out de novo without the use of a template.

[0031] Enzymatic DNA synthesis can involve a template, such as a DNA template.

[0032] Enzymatic DNA synthesis can be carried out in a reaction mixture containing the components described herein.

[0033] Alternatively, a cell-free method for synthesizing DNA is provided, the method comprising contacting a DNA template with at least one polymerase to form a reaction mixture in the presence of one or more nucleotides in salt form, wherein the nucleotides are present at a concentration of at least 10 mM and are: (a) In the form of a salt having a single monovalent cation, the ionic radius of which is greater than that of a sodium ion, or (b) A salt having two or more different monovalent cations, wherein at least one cation has an ionic radius greater than that of a sodium ion.

[0034] Alternatively, the nucleotide salts may comprise not only monovalent cations of sodium or lithium, but a significant proportion of nucleotide salts comprising cations with ionic radii greater than that of sodium ions. Thus, a cell-free method for synthesizing DNA is provided, comprising contacting a DNA template with at least one polymerase to form a reaction mixture in the presence of one or more nucleotides in the form of salts having monovalent cations, wherein the nucleotides are present at a concentration of at least 10 mM and are not limited to sodium or lithium.

[0035] Preferably, when referring to the concentration of a nucleotide or nucleotide salt, this refers to the concentration of the nucleotide (or its salt) at the start of the method, i.e., the initial or initial concentration of the nucleotide (or nucleotide salt). Therefore, it is the concentration after addition to the reaction mixture. It should be understood that other components may be added during the process of the method; such addition can dilute the concentration of the nucleotide / nucleotide salt unless additional nucleotides are provided to replenish it. Furthermore, as the nucleotide / nucleotide salt will be used or consumed by the method (i.e., the DNA synthesis reaction), its concentration will decrease as the method proceeds. In some embodiments, additional nucleotide / nucleotide salts may be added as the method proceeds to replenish the substrate used for the enzymatic reaction.

[0036] The inventors have surprisingly discovered that the need for divalent cations in synthesis is reduced when the nucleotide salt includes a monovalent cation with an ionic radius larger than that of a sodium ion. Conventionally, for example, magnesium (a divalent cation) is required in a minimum 1:1 ratio with the nucleotide salt in DNA synthesis reactions. This is because magnesium is needed at the active sites of certain polymerases; it can form complexes with nucleotides prior to integration and can also form its own salt with phosphate ions 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 included in DNA synthesis significantly lowers costs, and higher magnesium concentrations are associated with reduced accuracy in DNA synthesis.

[0037] Divalent cations may contain one or more metals selected from the following list: Mg 2+ Be 2+ Ca 2+ 、Sr 2+ Mn 2+ or Zn 2+ Mg is preferred 2+ or Mn 2+In the reaction mixture, the ratio of metal cation to nucleotide salt can be approximately 1:1. A ratio lower than 1:1 is desirable and preferred in DNA synthesis because a ratio higher than 1:1 can lead to some distortion in DNA synthesis. Divalent cations can be provided in the form of any suitable salt for enzymatic DNA synthesis.

[0038] Therefore, the present invention also relates to enzymatic DNA synthesis under conditions of reduced divalent cations, comprising using a nucleotide salt having one or more monovalent cations having an ionic radius greater than that of a sodium ion. The reduction in this case is compared to the same reaction in the presence of lithium or sodium ions in the nucleotide salt.

[0039] The inventors have surprisingly discovered that using nucleotide salts with alternative counterions (e.g., ammonium and cesium ions) in the method of the present invention reduces the need for buffers included in enzymatic DNA synthesis. This is also advantageous because it lowers the cost of the synthesis reaction and could be beneficial for DNA synthesis for therapeutic purposes.

[0040] Furthermore, regarding other components present, the method developed by the inventors herein can be carried out under a wide range of conditions. These conditions range from conventional buffer levels to the absence of additional buffers, effectively reacting with the desired components in water. The desired components may include DNA synthases (i.e., polymerases), nucleotide salts, and divalent cations (as salts), and optional other components selected from templates, denaturants, pyrophosphatases, or one or more primers, depending on the reaction conditions. These components can form a reaction mixture.

[0041] Therefore, it is advantageous to provide the method (i.e., the reaction mixture) with at least a certain proportion of nucleotides as salts of monovalent positive counterions (cations) having an ionic radius larger than that of sodium ions, because this surprisingly achieves the conversion of nucleotides into DNA with increased DNA yield and / or increased efficiency. These improvements can be compared to similar reaction mixtures in which all nucleotides are provided individually as conventional salts, for example, as lithium salts or sodium salts alone, or a mixture of both ions. Providing nucleotide salts different from those conventionally used has several additional surprising advantages, such as the ability to reduce the concentration of buffers in the reaction mixture (in some cases to zero), and the ability to reduce the need for divalent cationic cofactors (most notably magnesium) in the reaction mixture.

[0042] On one hand, there is the enzymatic DNA synthesis in template-directed methods. This template can be a DNA template. Template amplification is preferably via strand displacement. Template amplification is preferably isothermal, i.e., it does not require cycling between low and high temperatures for amplification. In this case, if necessary, the template can be denatured initially using heat, or it can be denatured chemically. However, once the template has been denatured, the temperature can be maintained within a range that does not affect the denaturation of the template and product, if appropriate, to allow any primers to enter between the double-stranded templates. Isothermal temperature conditions require that the reaction not be heated to a point that denatures the template and product (unlike PCR, which requires thermal cycling to denature the template and product). Typically, depending on the enzyme's preference, such reactions are carried out at a constant temperature. This temperature can be any temperature suitable for the enzyme.

[0043] Cell-free methods preferably involve replicating and amplifying a template via strand displacement. This synthesis releases single-stranded DNA, which can then be copied into double-stranded DNA using a polymerase. The term strand displacement describes the ability to replace downstream DNA encountered during the synthesis process, where the polymerase opens the double-stranded DNA to extend the nascent single strand. 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 replication helicase opens the double-stranded DNA and facilitates the advancement of the leader strand polymerase.

[0044] Independently, optional features of any aspect of the invention may include: the template may be circular. Strand substitution amplification of the DNA template may be performed by rolling circle amplification (RCA). The polymerase may be Phi29 or a variant thereof. DNA amplification may be isothermal, i.e., at a constant temperature. One or more primers may be random primers. A pair or a set of primers may be used. The synthesized DNA may comprise a tandem strand containing 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.

[0045] The amount of DNA that can be synthesized is equal to or greater than 3 g / L of the reaction mixture, particularly 16 g / L or higher, preferably up to 30 g / L or higher.

[0046] The amount of DNA that can be synthesized can exceed 60% of the maximum yield calculated for the reaction mixture. Preferably, the amount of DNA that can be synthesized can exceed 80% of the maximum yield calculated. The maximum yield calculated is based on the theoretical yield if all nucleotides are integrated into the product, which can be calculated by those skilled in the art.

[0047] The efficiency of DNA synthesis from nucleotides (or nucleotide salts) can be described as the percentage of nucleotides or their salts supplied to the reaction mixture that are successfully incorporated into the product during the reaction.

[0048] Cell-free methods require at least one nucleotide. One or more additional nucleotides may then be added. The nucleotide or additional nucleotide is a deoxyribonucleoside triphosphate (dNTP) or a derivative or modified form thereof. The nucleotide or additional nucleotide is one or more of the following: deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), and their derivatives. The nucleotide or additional nucleotide is provided as its salt. Each individual nucleotide salt may contain up to four monovalent cations to maintain electroneutrality. The nucleotide salt used in this method may include one or more monovalent cations, i.e., one or more monovalent cation species, and preferably, most (if not all) of said monovalent cations have an ionic radius greater than that of a sodium ion. It should be understood that these can dissociate in solution, thus contributing to the presence of cations in this method.

[0049] Preferably, in this method, i.e., in the reaction mixture, the concentration of the nucleotide or its salt can be greater than 10 mM and up to at least 100 mM. Such a concentration is important for producing DNA in higher yields, and in the two given concentrations, it can be up to 3 g / L to 30 g / L. Preferably, the concentration of the nucleotide or its salt is at the start of the method, i.e., the initial or initial concentration of the nucleotide or its salt in the reaction mixture, which also includes enzymes necessary for DNA synthesis. Subsequent addition of other components can reduce this concentration, and the use of DNA synthases therein will also decrease from the initial concentration. Those skilled in the art will understand how the concentration of the nucleotide / nucleotide salt is calculated during the preparation process based on the volume of other components and the stock solution / powder of nucleotide salts used.

[0050] The terms nucleotide and nucleotide salt are used interchangeably in the art because all nucleotides are inherently provided as salts.

[0051] This method can be a batch method or a continuous flow method. The batch can be a closed batch (i.e., all reaction components are provided at the start of DNA synthesis), or additional components can be provided to the reaction as needed during the process, as described in, for example, WO2016 / 034849, which is incorporated herein by reference. If additional addition is required, this will dilute the concentration of nucleotides or nucleotide salts unless additional nucleotide salts are added to replenish that concentration.

[0052] The inventors of this application have discovered that each different counterion can add specific characteristics to an enzymatic DNA synthesis reaction. For example, using a nucleotide salt containing cesium ions results in enzymatic DNA synthesis in the presence of reduced magnesium levels. Furthermore, the use of ammonium ions in the nucleotide salt has resulted in the use of some high concentrations of nucleotides, and examples show DNA synthesis at a nucleotide concentration of 80 mM.

[0053] The inventors had not previously recognized the use of several of these cations in nucleotide salts due to their lack of immediate commercial availability. These nucleotide salts can be custom-ordered from nucleotide manufacturers if needed.

[0054] Therefore, the use of nucleotide salts containing any of cesium, ammonium, ammonium derivatives, or rubidium cations in enzymatic cell-free DNA synthesis is part of this invention.

[0055] Enzymatic cell-free DNA synthesis can be performed in the presence of low levels of divalent cations, less than about 1:1, preferably with a ratio of divalent cation to nucleotide of 0.2:1 to 0.8:1, and more preferably 0.2:1 to 0.5:1. These ions are counterions in nucleotide salts.

[0056] Enzymatic cell-free DNA synthesis can be performed using these ions in minimal buffering agents, without the addition of other salts or washing agents that have been shown to enhance DNA synthesis or promote primer binding. Minimal buffering agents may contain pH-stabilizing reagents (buffers). Minimal buffering agents may contain very small amounts of cations, provided by the presence of chemicals used to denature the template (e.g., sodium hydroxide, potassium hydroxide, or ammonium hydroxide). The ions are counterions in nucleotide salts.

[0057] If low levels of magnesium ions are required in enzymatic DNA synthesis, the inventors have discovered that reliable nucleotides for such synthesis are nucleotide salts containing cesium ions.

[0058] Therefore, the present invention provides an enzymatic cell-free synthesis of DNA in which the ratio of divalent ions to nucleotides needs to be maintained at 0.5:1 or lower, the method comprising using a nucleotide salt containing cesium ions.

[0059] Other advantages are described below. Attached Figure Description

[0060] The invention will now be further described with reference to exemplary embodiments and accompanying drawings, wherein: Figure 1Figures A through 1E illustrate the results obtained through experiments using different initial concentrations of nucleotide salts with different counterions and different initial / starting concentrations of magnesium ions (as MgCl2) in the DNA synthesis reaction. Each figure shows the raw DNA yield (g / L) obtained versus the theoretical DNA yield (g / L) corresponding to the total initial / starting nucleotide salt concentration (mM). On all figures, dashed lines indicate a conversion efficiency of 80% of the nucleotide salts to DNA, and solid lines indicate a conversion efficiency of 100%. Figure 1 A describes the DNA synthesis results obtained using lithium-dNTPs; Figure 1 B is the result obtained using sodium-dNTPs. Figure 1 C was obtained using potassium-dNTPs. Figure 1 D was obtained using ammonium-dNTPs. Figure 1 E is the result when using cesium-dNTP; Figure 2 This is a graph showing data from DNA synthesis experiments, specifically a graph showing the dNTP salt concentration (mM) for maximum raw DNA yield relative to different concentrations of magnesium ions. Results are shown using nucleotide salts with lithium, sodium, potassium, ammonium, and cesium as counterions. The graph is divided into three sections, highlighting results where the magnesium ion to nucleotide salt ratio is less than 0.5, the sections with ratios of 0.5 and 1, and the final section with ratios exceeding 1. Thresholds for these sections are also shown—dashed lines represent a 0.5:1 magnesium ion to nucleotide (dNTP) ratio, and solid lines represent a 1:1 ratio. Figure 3 This is a graph showing data obtained from DNA synthesis experiments. In this example, DNA yields were measured for various DNA synthesis reactions using nucleotide salts with various counterions at fixed starting concentrations and with increasing concentrations of magnesium chloride. The graph shows the raw DNA yield (g / L) for all tested nucleotide salts relative to magnesium chloride concentrations. Figure 4 This is a graph showing data from a DNA synthesis reaction using rolling circle amplification with varying initial concentrations of nucleotide salts in a minimal buffer. The graph plots raw DNA yield (g / L) relative to the initial / starting dNTP salt concentration (mM). Figure 5This is a diagram of a DNA synthesis experiment using rolling circle amplification with a DNA template. During the experiment, the presence of additional monovalent cations in the reaction mixture was tested to determine if it affected the DNA synthesis reaction. In this experiment, nucleotide ammonium salts were used, and the monovalent cation chloride salts shown were also included in the reaction mixture. The initial ratio of dNTP ammonium salt to monovalent chloride salt was 1:4; this is because a monovalent cation is provided for each ammonium ion present on the dNTP (of which there are four). Therefore, the initial ratio of ammonium ions (on the dNTP salt) to monovalent cations was 1:1. The initial magnesium concentrations were also different, at 5 mM, 10 mM, 20 mM, and 40 mM, corresponding to 17.5 mM, 25 mM, 35 mM, and 50 mM concentrations of the ammonium counterion dNTPs shown in the diagram, respectively. The raw DNA yield of ammonium counterion dNTPs (NH4-dNTPs) at the indicated concentrations was plotted in the presence of monovalent cations (including lithium, sodium, potassium, ammonium, and cesium) counterion chloride salts, while controls were prepared without additional salts.

[0061] Figure 6 Figures A and 6B are the results of several pH assays comparing the pH of reaction mixtures lacking polymerase, template, and primers at various initial concentrations of nucleotide salts. Variable initial magnesium chloride (MgCl2) concentrations were used. The figure shows the pH measured relative to the nucleotide salt concentration at the stated initial magnesium chloride concentration. No DNA synthesis occurred. Figure 6 A shows a data graph of cesium-dNTPs. Figure 6 B shows a data graph of ammonium-dNTPs; and

[0062] Figure 7 The plasmid diagram proTLx-K B5X4 LUX ST(AT) used in the examples shows the processing site (TelRL), Luc 2 reporter gene, kanamycin resistance gene, CMV promoter, and pUC ori. Detailed Implementation

[0063] This invention relates to a cell-free method for large-scale DNA synthesis. The method of this invention allows for high-throughput DNA synthesis.

[0064] The deoxyribonucleic acid (DNA) synthesized according to the present invention can be any DNA molecule. The DNA can be single-stranded or double-stranded. The DNA can be linear. The DNA can be processed to form loops, particularly microloops, single-stranded closed loops, double-stranded closed loops, double-stranded open loops, or closed linear double-stranded DNA. The DNA can be permitted to form or be processed to form specific secondary structures, such as, but not limited to, hairpin loops (stem loops), imperfect hairpin loops, pseudo-junctions, or any of various types of double helices (A-DNA, B-DNA, or Z-DNA). The DNA can also form hairpin and aptamer structures.

[0065] The synthesized DNA can have any suitable length. Using the method of the present invention, a length of up to or greater than 77 kilobases is possible. More particularly, the DNA length that can be synthesized according to the method of the present invention can 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 can be 100 kilobases to more than 77 kilobases, 500 kilobases to 60 kilobases, 200 kilobases to 20 kilobases, more preferably 200 kilobases to 15 kilobases, and most preferably 2 kilobases to 15 kilobases.

[0066] The amount of DNA synthesized according to the method of the present invention can exceed 3 g / L. Preferably, the amount of DNA synthesized is greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 g / L or higher. A preferred amount of synthesized DNA is 5 g / L. The amount of DNA produced can be described as industrial or commercial quantities produced on a large scale or in large quantities. The DNA produced by the method of the present invention can be consistent in quality (i.e., DNA length and sequence). Therefore, this method is suitable for large-scale DNA synthesis. The method can be consistent in terms of the accuracy of synthesis.

[0067] Alternatively, the amount of DNA produced in the synthesis reaction can be compared to the theoretical maximum yield, which is achieved if 100% nucleotides are integrated into the synthesized DNA. The method of the present invention not only increases the total yield obtained but also improves the efficiency of the method, meaning that more provided nucleotides are integrated into the synthesized DNA product compared to previous methods. The yield obtainable by the method of the present invention exceeds 50% of the theoretical maximum, up to and exceeding 90% of the theoretical maximum. Therefore, the percentage of the theoretical maximum yield achieved by the method of the present invention includes 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% or higher. Conventionally, using commercially available nucleotide salts, the yield achieved is disappointing due to the potential ionic effects that inhibit the method.

[0068] DNA is synthesized via an enzymatic reaction. This enzymatic synthesis can involve the use of any DNA synthase, most obviously polymerases or modified polymerases. These will be discussed further below. DNA synthesis can be de novo and does not require a template. Enzymatic synthesis can also require the use of a template for DNA synthesis. The template can be any suitable nucleic acid, depending on the polymerase, but is preferably a DNA template.

[0069] The template can be any suitable template, providing instructions for DNA synthesis simply by including a specific sequence. The template can be single-stranded (ss) or double-stranded (ds). The template can be linear or circular. The template can include natural, artificial, or modified bases or mixtures thereof.

[0070] Templates can contain any natural or artificial sequence.

[0071] The template can have any suitable length. In particular, the template can be up to 60 kilobases, or up to 50 kilobases, or up to 40 kilobases, or up to 30 kilobases. Preferably, the DNA template can 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.

[0072] The template can be provided in a quantity sufficient for use in that method by any method known in the art. For example, the template can be generated by PCR.

[0073] The method can amplify all or selected portions of the template.

[0074] The template may contain an expression sequence. The DNA can be used for expression in cells (i.e., cells transfected in vitro or in vivo) or for expression in cell-free systems (i.e., protein synthesis). The expression sequence can be used for therapeutic purposes, i.e., gene therapy for DNA vaccines. The expression sequence can be a gene that encodes a DNA vaccine, therapeutic protein, etc. The sequence may contain a sequence transcribed into active RNA (i.e., small interfering RNA molecules (siRNA)).

[0075] If necessary, the template can be contacted with at least one polymerase, as described below.

[0076] Enzymatic DNA synthesis reactions may require at least one DNA synthase. 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 may be used. The polymerase may be any suitable polymerase from any family of polymerases to synthesize a polymer of DNA. The polymerase may be a DNA polymerase. Any DNA polymerase may be used, including any commercially available DNA polymerase. Two, three, four, five, or more different DNA polymerases may be used, for example, one providing proofreading activity while one or more others do not. DNA polymerases with different mechanisms may be used, such as strand substitution polymerases and DNA polymerases that replicate DNA by other methods. A suitable example of a DNA polymerase that does not have strand substitution activity is T4 DNA polymerase. Template-independent polymerases, such as terminal transferases, may be used.

[0077] Modified polymerases can also be used. These can be engineered to alter their properties, such as eliminating their dependence on the template, changing their temperature dependence, or stabilizing the enzyme for in vitro use.

[0078] The polymerase can be highly stable so that prolonged incubation under processing conditions does not substantially reduce its activity. Therefore, the enzyme preferably has a long half-life under a range of process conditions, including but not limited to temperature and pH. It is also preferred that the polymerase possesses one or more features suitable for the manufacturing method. The polymerase preferably exhibits high accuracy, for example, by possessing proofreading activity. Furthermore, it is preferred that the polymerase displays high sustained synthetic capacity, high strand substitution activity, and K+ for dNTPs and DNA. m Low. The polymerase may be able to use circular and / or linear DNA as a template. The polymerase may be able to use dsDNA or ssDNA as a template. Preferably, the polymerase does not exhibit DNA exonuclease activity unrelated to its proofreading activity.

[0079] Those skilled in the art can use commercially available polymerases (e.g., Phi29 (New England Biolabs, Inc., Ipswich, MA, US), Deep Vent® (New England Biolabs, Inc.), and Bacillus stearothermophilus (…) Bacillus stearothermophilus To determine whether a given polymerase exhibits the characteristics defined above, the properties of Bst DNA polymerase I (New England Biolabs, Inc.), the Klenow fragment of DNA polymerase I (New England Biolabs, Inc.), M-MuLV reverse transcriptase (New England Biolabs, Inc.), VentR® (exo-minus) DNA polymerase (New England Biolabs, Inc.), VentR® DNA polymerase (New England Biolabs, Inc.), Deep Vent® (exo-) DNA polymerase (New England Biolabs, Inc.), and Bst DNA polymerase large fragment (New England Biolabs, Inc.) are compared. When referring to high sustained synthetic capacity, this typically refers to the average number of nucleotides added by the polymerase during each binding / dissociation with the template, i.e., the length of the nascent extension obtained from a single binding event.

[0080] Chain displacement polymerases are preferred. Preferred chain displacement polymerases are Phi29, Deep Vent, and Bst DNA polymerase I, or variants of any one of them. "Chain displacement" describes the polymerase's ability to displace the 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. Therefore, during chain displacement replication, the newly replicated strand is displaced to make way for the polymerase to replicate another complementary strand. The amplification reaction is initiated when the 3' free end of a primer or single-stranded template anneals to the complementary sequence on the template (both are priming events). As DNA synthesis proceeds, if it encounters another primer or other strand annealed to the template, the polymerase displaces it and continues its strand extension. Chain displacement can release single-stranded DNA, which can then serve as template for further priming events. Priming of the newly released DNA can lead to hyperbranching and high product yields. It should be understood that the difference between chain displacement amplification methods and PCR-based methods is that denaturation cycles are not essential for efficient DNA amplification because double-stranded DNA is not an obstacle to the continued synthesis of new DNA strands. If primers are used, strand displacement amplification may require only an initial heating cycle to denature the initial template (if it is double-stranded), thereby annealing the primers to their binding sites. After this, since no further heating or cooling is needed, the amplification can be described as isothermal. In contrast, PCR methods require denaturation cycles (i.e., raising the temperature to 94 degrees Celsius or higher) during the amplification process to unwind the double-stranded DNA and provide new single-stranded templates. During strand displacement, a polymerase displaces the already synthesized DNA strands. Furthermore, it uses the newly synthesized DNA as a template, ensuring rapid DNA amplification.

[0081] The strand substitution polymerase used in the method of the present invention preferably has a sustained synthetic capacity 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 substitution DNA polymerase has a sustained synthetic capacity comparable to or greater than that of the phi29 DNA polymerase.

[0082] Therefore, strand substitution replication is preferred. During strand substitution replication, the template is amplified by replacing the already replicated strand (which has been synthesized by polymerase) with another strand (which can be the original complementary strand of the double-stranded template or a newly synthesized complementary strand, the latter being synthesized by polymerase acting on a prior primer annealed to the template). Thus, template amplification can occur by replacing the already replicated strand with strand substitution replication of another strand. This method can be described as strand substitution amplification or strand substitution replication.

[0083] The preferred method for strand substitution replication is loop-mediated isothermal amplification (LAMP). LAMP typically uses 4-6 primers to recognize 6-8 distinct regions of the template DNA. In short, DNA polymerase of the substitution strand initiates synthesis, and two of the primers form a loop structure to facilitate subsequent amplification cycles. The inner primer, containing the sense and antisense sequences of the target DNA, initiates LAMP. The subsequent strand substitution DNA synthesis, initiated by the outer primer, releases single-stranded DNA. This serves as a template for DNA synthesis initiated by a second inner primer hybridizing to the other end of the target DNA, producing a stem-loop DNA structure. In subsequent LAMP cycles, one inner primer hybridizes to the loop on the product and initiates substitution DNA synthesis, resulting in the original stem-loop DNA and a new stem-loop DNA with a stem twice the length. Modified LAMP procedures can also be used where fewer inner primers are required.

[0084] The preferred method for strand substitution replication is rolling circle amplification (RCA). The term RCA describes the ability of an RCA-type polymerase to continuously extend hybridization primers around a circular DNA template strand. This results in the formation of a linear single-stranded product of amplified DNA with multiple repeats. The sequence of the circular template (single unit) is repeated multiple times within the linear product. For a circular template, the initial product of strand substitution amplification is a single-stranded polyp, which is 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 substitution events, resulting in the formation of a polyp double-stranded DNA product, again containing multiple repeats of amplified DNA. Thus, multiple copies of each amplified “single unit” of DNA are present in the polyp double-stranded DNA product. RCA polymerase is particularly preferred for use in the method of the present invention. The product of the RCA-type strand substitution replication method may require processing to release the single-unit DNA. This is desirable if a single unit of DNA is desired. Typical strand substitution conditions using Phi29 DNA polymerase include high levels of magnesium ions, such as 10 mM magnesium (usually a chloride salt), and 0.2 to 4 mM nucleotides.

[0085] To allow amplification, enzymatic DNA synthesis may require one or more primers, depending on several aspects. If no template is used, the primers will provide the starting point for DNA synthesis and are designed to initiate the synthetic reaction. If a template is used, the primers can be nonspecific (i.e., random sequences) or specific to one or more sequences contained within the template. Alternatively, a primase can be provided to generate primers de novo. If primers have random sequences, they allow nonspecific initiation at any site on the template. This allows for efficient amplification through multiple initiation reactions from each template strand. Examples of random primers are hexamers, heptamers, octamers, nonamers, decamers, or longer sequences, such as sequences of 12, 15, 18, 20, or 30 nucleotides in length. Random primers can be 6 to 30, 8 to 30, or 12 to 30 nucleotides in length. Random primers are often provided as mixtures of oligonucleotides, representing all potential combinations of, for example, hexamers, heptamers, octamers, or nonamers in the template.

[0086] In one implementation, the primers, or one or more primers, are specific. This means they have sequences complementary to the sequence in the template from which amplification is expected to be initiated. In this implementation, a pair of primers can be used to specifically amplify a portion of the DNA template located within the two primer binding sites. Alternatively, a single specific primer can be used. A set of primers can also be used.

[0087] Primers can be any nucleic acid composition. Primers can be unlabeled or may contain one or more labels, such as radionuclides or fluorescent dyes. Primers may also contain chemically modified nucleotides. For example, primers can be capped to prevent the initiation of DNA synthesis until the cap is removed, i.e., by chemical or physical means. Primer length / sequence is typically selected based on temperature considerations, i.e., the temperature at which it binds to the template during the amplification step.

[0088] In some respects, the contact between the template and the polymerase and one or more primers can be performed under conditions that promote primer annealing to the template. These conditions include the presence of a single-stranded nucleic acid that allows primer hybridization. Conventionally, the conditions also include a temperature and buffer that allow the primers to anneal to the template. Appropriate annealing / hybridization conditions can be selected based on the properties of the primers. An example of conventional annealing conditions that can be used in this invention includes a buffer containing 30 mM Tris-HCl pH 7.5, 20 mM KCl, and 8 mM MgCl2. However, the inventors have described conditions herein with reduced buffer and divalent metal ion components that still allow primer binding, and these will be discussed further below. Annealing can be performed after denaturation using heat, followed by gradual cooling to the desired reaction temperature.

[0089] However, amplification can also be performed using strand substitution replication without primers, thus eliminating the need for hybridization and primer extension. Instead, the single-stranded template self-initiates by forming a hairpin with a free 3'-end that can be used for extension. The remaining steps of amplification remain the same.

[0090] The template and / or polymerase also contact the nucleotide as a nucleotide salt. The combination of DNA template, polymerase, and nucleotide salt can be described as forming a reaction mixture. The reaction mixture may also contain one or more primers or primases. The reaction mixture may also independently include one or more divalent metal cations. The reaction mixture may further contain a chemical denaturing agent. Such a denaturing agent may be potassium hydroxide, ammonium hydroxide, or sodium hydroxide. The reaction mixture may further contain additional enzymes, such as helicases or pyrophosphatases. The reaction mixture may contain a pH buffer, and in some respects, it may not contain a pH buffer.

[0091] A nucleotide is a monomer or single unit of nucleic acid, and it consists of a nitrogenous base, a pentose sugar (ribose or deoxyribose), and at least one phosphate group. Any suitable nucleotide can be used.

[0092] Nucleotides exist as salts of monovalent cations. A monovalent cation is an ionic substance with a single positive charge, and therefore typically there are at most four in a nucleotide salt. Preferably, the ionic radius of a monovalent cation is greater than that of a sodium ion. The ionic radius is the radius of an ion in an ionic crystal structure. Ionic radii are typically given in picometers (pm) or angstroms (Å). The ionic radius is not a fixed property of a given ion, but varies with various parameters, including coordination number and spin state. However, ionic radius values ​​are sufficiently diverse to allow identification of periodic trends in atomic ions, with the ionic radius increasing as the periodic table group decreases. For the same ion, the ionic radius increases with increasing coordination number, and an ion in a low-spin state will be smaller than the same ion in a high-spin state. Generally, the ionic radius decreases with increasing positive charge. Therefore, when referring to the ionic radius herein, it can be any possible ionic radius of the ion. Exemplary ionic radii are listed in Table 6.

[0093] Nucleotides can include salts of monovalent metal ions, including but not limited to alkali metals (Group 1): lithium (Li). + ), sodium (Na + ), potassium (K) + ), Rubidium (Rb + ), Cesium (Cs) + ) or francium (Fr + Alternatively or additionally, the monovalent metal ion can be a transition metal (Group 11): copper (Cu). + ), silver (Ag) + ), gold (Au)+ ) or 𬬭 (Rg + Alkali metals are preferred, therefore the preferred counter ion could be potassium (K). + ), Rubidium (Rb + ), Cesium (Cs) + ) or francium (Fr + ).

[0094] Nucleotides can include salts of polyatomic monovalent ions. A polyatomic ion is an ion containing more than one atom. This distinguishes polyatomic ions from monatomic ions containing only one atom. Exemplary monovalent polyatomic cations include ammonium (NH4)2. + ) and hydrated hydrogen ions (H3O) + Ammonium is particularly preferred. Under all conditions, the ionic radius of ammonium is greater than that of sodium. Derivatives of ammonium are also included, an exemplary list of which includes: monoalkylammonium, dialkylammonium, trialkylammonium, choline, quaternary ammonium, and imidazoline. Those skilled in the art will recognize other derivatives of ammonium carrying a single positive charge, which are suitable as counterions on nucleotide salts.

[0095] Nitrogenous bases can be adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Nitrogenous bases can also be modified bases, such as 5-methylcytosine (m5C), pseudouridine (Ψ), dihydrouridine (D), inosine (I), and 7-methylguanosine (m7G). Nitrogenous bases can further be artificial bases. The concentration of the nucleotide salt can include any combination of various nitrogenous bases.

[0096] The preferred pentose sugar is deoxyribose, which makes the nucleotide a deoxynucleotide.

[0097] Nucleotides can be in the form of deoxynucleoside triphosphates (referred to as dNTPs). This is a preferred embodiment of the invention. Suitable dNTPs may include dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dTTP (deoxythymidine triphosphate), dUTP (deoxyuridine triphosphate), dCTP (deoxycytidine triphosphate), dITP (deoxyinosine triphosphate), dXTP (deoxyxanthoside triphosphate), and their derivatives and modified forms. Preferably, the dNTPs comprise one or more of dATP, dGTP, dTTP, or dCTP, or their modified forms or derivatives. A mixture of dATP, dGTP, dTTP, and dCTP, or their modified forms, is preferred. Any suitable ratio of these dNTPs can be used as needed for the reaction.

[0098] Nucleotides or nucleotide salts may be in solution form or may be required to be provided as a solid, such as a powder. Nucleotides or nucleotide salts may contain modified nucleotides. Nucleotides or nucleotide salts may be provided as a mixture of one or more suitable bases, preferably one or more of adenine (A), guanine (G), thymine (T), and cytosine (C). Two, three, or preferably all four nucleotides (A, G, T, and C) may be used in the method of DNA synthesis. These nucleotides or nucleotide salts may all be present in substantially equal amounts, or, depending on the nature of the DNA to be synthesized, one or two may be provided in greater quantities.

[0099] Nucleotides can be all natural nucleotides (i.e., unmodified), they can be modified nucleotides that function like natural nucleotides and are biologically active (i.e., LNA nucleotides – locked nucleic acids), they can be modified and biologically inactive, or they can 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) can be provided in one or more forms, i.e., unmodified and modified, or biologically active and biologically inactive. All of these nucleotides are capable of forming suitable salts.

[0100] In one aspect of the invention, the nucleotide or nucleotide salt is present at a concentration of at least 10 mM. According to this aspect, the nucleotide or nucleotide salt may be present in the reaction mixture at concentrations greater than 10 mM, greater than 15 mM, greater than 20 mM, 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. Such concentrations are given as the concentration of the nucleotide salt at the start of the method. Concentrations are given after the addition of the nucleotide / nucleotide salt, wherein it may be added to the reaction mixture. The nucleotide salt may be any suitable mixture of nucleotide salts having different nitrogenous bases. The concentration applies to the sum of the nucleotide salts present at the start of the method, regardless of their composition. Therefore, for example, a 10 mM concentration of nucleotide salt can be any mixture of dCTP, dATP, dGTP, and dTTP with appropriate monovalent cations as counterions.

[0101] It will be understood that nucleotides provided as salts can dissociate in water and other solvents to form anionic nucleotide entities and cations.

[0102] The formation of nucleotide salts by counterions with ionic radii larger than that of sodium ions is a preferred aspect of any aspect of the invention. However, polymerases or DNA synthases may be tolerant of certain concentrations of lithium and / or sodium nucleotide salts. Therefore, a portion of the nucleotide salt in the method of the invention may be included, wherein the counterions are sodium and / or lithium. This portion is preferably less than 25%, optionally 20%, 15%, 10%, 5%, 1%, or less. Polymerases or DNA synthases may also be tolerant of sodium and / or lithium from other sources, such as denaturants. Preferably, the total concentration of lithium ions in the reaction mixture does not exceed 15 mM, preferably not more than 10 mM, and even more preferably not more than 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less. Since lithium appears to be more inhibitory, it is preferable to substantially exclude this ion from the reaction mixture. In the case of sodium ions, the presence of sodium ions can be tolerated because sodium hydroxide is typically used as a denaturant.

[0103] Therefore, the nucleotide salts used in the methods of the present invention can comprise mixtures of different nucleotide salts, such as mixtures of potassium-nucleotide salts and cesium-nucleotide salts. Many different salts can be used. Preferably, at least 75% of the salt has a counterion with an ionic radius greater than that of a sodium ion, optionally 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more. It is desirable to use mixtures of different salts to maximize DNA yield and take advantage of the different properties of various counterions. Alternatively, a cell-free method for synthesizing DNA is provided, comprising contacting a DNA template with at least one polymerase in the presence of one or more nucleotide salts to form a reaction mixture, wherein the nucleotides are in the form of two or more salts, each containing a different monovalent cation, wherein at least one cation has an ionic radius greater than that of a sodium ion. Therefore, two or more different nucleotide salts can be used in the methods of the present invention, the salts differing due to the use of different counterions. It is preferable that all salts use counterions with ionic radii greater than that of sodium ions.

[0104] Enzymatic DNA synthesis can be maintained under conditions that promote DNA synthesis, depending on the specific method chosen.

[0105] Template amplification via strand substitution is preferred. Preferably, the conditions promote amplification by replacing the replicated strand with the template through strand substitution replication of another strand. Conditions include using any temperature that allows DNA amplification, typically in the range of 20 to 90 degrees Celsius. Preferred temperature ranges may be about 20 to about 40 degrees Celsius or about 25 to about 35 degrees Celsius. For LAMP amplification, a preferred temperature is about 50 to about 70 degrees Celsius.

[0106] Typically, the appropriate temperature for enzymatic DNA synthesis is selected based on the temperature at which a specific polymerase exhibits optimal activity. This information is generally available and constitutes part of the ordinary knowledge of those skilled in the art. For example, when using phi29 DNA polymerase, a suitable temperature range is about 25 to about 35 degrees Celsius, preferably about 30 degrees Celsius. However, the thermally stable phi29 can be operated at higher, constant temperatures. Those skilled in the art can generally identify suitable temperatures for efficient amplification according to the method of the invention. For example, the method can be performed within a certain temperature range, and the yield of amplified DNA can be monitored to identify the optimal temperature range for a given polymerase. Amplification can be performed at a constant temperature, and preferably the method is isothermal. Because strand displacement amplification is preferred, it is not required to change the temperature to separate the DNA strands. Therefore, the method can be isothermal.

[0107] Other conditions generally considered to promote DNA synthesis include the presence of a suitable buffer / pH and other factors required for enzyme performance or stability. Suitable conventional conditions include any conditions known in the art for providing polymerase activity.

[0108] For example, the pH of the reaction mixture can be in the range of 3 to 10, preferably 5 to 8 or about 7, such as about 7.5. The pH can be maintained in this range by using one or more buffers. Such buffers include, but are not limited to, MES, Bis-Tris, ADA, ACES, PIPES, MOBS, MOPS, MOPSO, Bis-Tris propane, BES, TES, HEPES, DIPSO, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, tris(hydroxymethyl)methylglycine, Gly-Gly, N,N-dihydroxyethylglycine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS, phosphates, citrate-sodium hydrogen phosphate, citrate-sodium citrate, sodium acetate-acetic acid, imidazole, and sodium carbonate-sodium bicarbonate.

[0109] Buffer solutions are typically defined as mixtures of reaction components. They typically include a buffer to maintain a stable pH; one or more additional salts consisting of cationic and anionic substances, such as sodium chloride or potassium chloride; and / or a detergent (e.g., Triton-X-100) to ensure optimal enzyme activity or stability. Minimal buffers consist only of buffering reagents, without providing additional salts or detergents, provided that small amounts of cationic substances may be present in DNA synthesis requiring chemical denaturation. Surprisingly, the use of higher concentrations of nucleotide salts in the methods of this invention allows for the use of these minimal buffers.

[0110] A "buffer-free" system lacks the provided or defined pH buffer and additional salts or detergents in the mixture of reaction components. This "buffer-free" system contains only the reaction components required for DNA synthesis and cationic substances that provide counterions for chemical denaturation or simply as nucleotide salts. Therefore, no additional ions are added in this system besides those used for specific purposes in the DNA synthesis reaction. The counterions provided with the nucleotides (as salts) are used to stabilize the nucleotides prior to their use in this method.

[0111] While applying heat (exposure to 95°C for several minutes) is used to denature double-stranded DNA, other methods more suitable for DNA synthesis can be used. Double-stranded DNA can be readily denatured by exposure to high or low pH environments, or in the absence of cations or in very low concentrations (e.g., in deionized water). Polymerases require short oligonucleotide primer sequences to bind to single-stranded regions of the DNA template to initiate its replication. The stability of this interaction, and therefore the efficiency of DNA synthesis, can be particularly affected by metal cations, especially divalent cations such as Mg. 2+ The effect of ion concentration can be considered an indispensable part of this method.

[0112] Enzymatic DNA synthesis may also require divalent metal ions. This method may include using salts of divalent metal ions: magnesium (Mg²⁺). 2+ ), manganese (Mn) 2+ ), calcium (Ca 2+ ), beryllium (Be 2+ ), Zinc (Zn) 2+ ) and Strontium (Sr 2+ The most commonly used divalent ions in DNA synthesis are magnesium or manganese.

[0113] Enzymatic DNA synthesis can be carried out at concentrations of divalent metal ions that are lower than previously thought possible. Traditionally, a divalent cation to nucleotide ratio of at most 2:1 has been considered desirable or optimal, and as the data in the examples show, this is particularly applicable to nucleotide salts containing lithium ions, especially when lithium ions are the predominant form used. However, if alternative ions are used in these salts, the demand for divalent ions (particularly magnesium) decreases dramatically, resulting in an ion to nucleotide salt ratio of approximately 1.5:1 or approximately 1:1 or lower. Even results with magnesium to nucleotide salt ratios of 0.2:1 have been obtained using nucleotide salts containing cesium. These ratios are particularly pronounced at higher nucleotide salt concentrations (i.e., 20 mM or higher). Therefore, the present invention also relates to DNA synthesis in which the ratio of magnesium ions to nucleotide salts is 1:1 or lower, characterized in that the nucleotide salts contain counterions with ionic radii greater than those of sodium ions, and the concentration of the nucleotide salts 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.

[0114] During synthesis, the polymerase releases pyrophosphate from the nucleotides integrated into the growing DNA strand. Pyrophosphate has a binding affinity for magnesium ions similar to that of nucleoside triphosphates, thus the process does not release free magnesium ions. Using a high initial concentration of nucleotides during synthesis results in a reduced level of free magnesium ions. Because these ions are likely essential for the polymerase's catalytic activity, suboptimal levels caused by interactions with phosphate or phosphate groups are generally considered detrimental to efficient amplification. Therefore, a sufficiently high, and thus excessive, magnesium ion concentration is considered crucial for DNA yield and amplification. Thus, the ability to reduce magnesium levels while maintaining yield is an exciting improvement relative to existing techniques.

[0115] Therefore, the present invention provides an enzymatic DNA synthesis performed under conditions of reduced ratio of divalent cations to dNTPs, comprising using a nucleotide salt of one or more monovalent cations having an ionic radius greater than that of a sodium ion.

[0116] The effect is particularly pronounced when using nucleotide salts containing ammonium and cesium or a mixture thereof.

[0117] In some respects, the reaction mixture may also include a detergent. Examples of suitable detergents include Triton X-100. TM Tween 20 TMAnd derivatives of any of these. The reaction mixture may also include stabilizers. Any suitable stabilizer can be used, especially bovine serum albumin (BSA) and other stabilizing proteins. Reaction conditions can also be improved by adding reagents that facilitate DNA relaxation and template denaturation. Such reagents include, for example, dimethyl sulfoxide (DMSO), formamide, glycerol, and betaine. The reaction mixture may also include DNA concentrators. Such reagents include, for example, polyethylene glycol or cationic lipids or cationic polymers.

[0118] However, in some implementations, such as in a minimum buffer system or a buffer-free system, these components can be reduced or removed from the reaction mixture.

[0119] It should be understood that those skilled in the art can use these additional ingredients and conditions, based on their ordinary knowledge, to modify and optimize the synthesis conditions for the methods used in this invention. Similarly, the specific concentrations of particular reagents can be selected based on previous examples in the art, and further optimized based on ordinary knowledge.

[0120] 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, and 1 mM dNTP. The preferred reaction buffer used in the RCA amplification of this invention is 30 mM Tris-HCl, pH 7.9, 30 mM KCl, 7.5 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, and 2 mM dNTP. This buffer is particularly suitable for use with Phi29 DNA polymerase.

[0121] Suitable reaction buffers for use with the nucleotide salts of the present invention are 30 mM Tris HCl, pH 7.9, 5 mM (NH4)2SO4, and 30 mM KCl. In some cases, enzymatic DNA synthesis can be carried out in water (“buffer-free”).

[0122] Enzymatic DNA synthesis may also involve the use of one or more additional proteins. The DNA template can be amplified in the presence of at least one pyrophosphatase, such as a yeast inorganic pyrophosphatase. Two, three, four, five, or more different pyrophosphatases can be used. These enzymes are capable of degrading pyrophosphate produced by polymerases during strand replication. Accumulation of pyrophosphate in the reaction can lead to inhibition of DNA polymerase and reduce the rate and efficiency of DNA amplification. Pyrophosphatases can break down pyrophosphate into non-inhibitory phosphate. An example of a suitable pyrophosphatase for the method of the present invention is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae Pyrophosphatase, which is commercially available from New England Biolabs, Inc.

[0123] Any single-stranded binding protein (SSBP) can be used in the method of this invention to stabilize single-stranded DNA. SSBPs are essential components of living cells and are involved in all processes involving ssDNA, such as DNA replication, repair, and recombination. In these processes, SSBPs bind to transiently formed ssDNA and help stabilize the ssDNA structure. An example of a suitable SSBP for use in the method of this invention is the T4 gene 32 protein, which is commercially available from New England Biolabs, Inc.

[0124] The yield of the reaction is related to the amount of DNA synthesized. The expected yield of the method according to the invention can exceed 3 g / L. Preferably, the amount of DNA synthesized is greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 g / L or higher. A preferred amount of DNA synthesized is 5 g / L. This invention increases the possible yield of enzymatically synthesized DNA. One objective of this invention is to increase the yield of cell-free enzymatic DNA synthesis methods, thereby enabling large-scale DNA synthesis in a cost-effective manner. This invention allows for the economical production / synthesis of DNA on an industrial scale using enzymatic methods catalyzed by DNA synthases or polymerases. The method of this invention allows for the efficient integration of nucleotides into the DNA product. It is believed that the method of this invention allows the reaction mixture to be scaled up to several liters, including tens of liters. Increased yield, productivity, or continuous synthetic capacity can be compared to the same reaction mixture in which all nucleotides are provided as conventional salts (sodium and / or lithium).

[0125] In one embodiment, the present invention relates to a method for enhancing DNA synthesis. This enhancement can be compared to a reaction mixture that is identical except that all nucleotide salts used are sodium or lithium, or mixtures thereof.

[0126] In one aspect, the present invention provides a cell-free method for synthesizing DNA, the method comprising contacting a DNA template with at least one polymerase to form a reaction mixture in the presence of one or more nucleotides in the form of salts having one or more monovalent cations, wherein the nucleotides are present at a concentration of at least 10 mM and the cations are not all sodium or lithium.

[0127] Alternatively, a cell-free method for synthesizing DNA includes contacting a DNA template with at least one polymerase in the presence of one or more nucleotides in salt form to form a reaction mixture, wherein the nucleotides are present at a concentration of at least 10 mM and are: (a) In the form of a salt having a single monovalent cation, wherein the ionic radius of the monovalent cation is greater than that of the sodium ion, or (b) A salt having two or more different monovalent cations, wherein at least one cation has an ionic radius greater than that of a sodium ion.

[0128] Preferably, the nucleotide concentration mentioned herein refers to the initial concentration of nucleotides at the start of the method, i.e., the initial concentration when the reaction mixture is formed.

[0129] The present invention also relates to a cell-free method for synthesizing DNA, the method comprising contacting a DNA template with at least one polymerase in the presence of one or more nucleotides, said nucleotides being in the form of salts containing sodium ions at a concentration of 10 to 20 mM, or up to 30 mM. The present invention provides a cell-free method for enzymatic synthesis of DNA, the method comprising using a nucleotide provided as a salt, said salt comprising a monovalent cation with an ionic radius greater than that of a sodium ion, preferably said nucleotide salt being provided or present at a concentration greater than 10 mM.

[0130] The present invention further provides enzymatic DNA synthesis under conditions where divalent cations, preferably magnesium, are reduced, including the use of nucleotide salts having one or more monovalent cations with ionic radii greater than that of sodium ions.

[0131] Alternatively, the present invention can be carried out using a nucleotide salt having an ionic radius greater than that of a potassium ion. Optionally, 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.

[0132] Nucleotides in salt form are also referred to as nucleotide salts in this paper.

[0133] The invention will now be described with reference to several non-limiting embodiments.

[0134] Example

[0135] Materials and methods

[0136] reagents

[0137] The following reagents were used in the provided examples: dNTP lithium salt, stock concentration 100 mM (Bioline) dNTP salts in sodium, potassium, cesium, and ammonium forms, with a stock concentration of 100 mM (contract synthesis). Phi29 DNA polymerase, stock concentration 2.4 g / L (internal production) Thermostable pyrophosphatase, stock concentration 2000 U / ml (Enzymatics) DNA primers, stock concentration 5 mM (Oligofactory) Plasmid template: ProTLx-K B5X4 LUX 15-0-15-10-15 AT-STEM The stock concentration is 0.1 g / L (internal production). Nuclease-free water (Sigma Aldrich) Magnesium chloride, stock concentration 2M (Sigma Aldrich) Tris-base (Thermo Fisher Scientific) Tris-HCl (Sigma Aldrich) NaCl (Sigma Aldrich) EDTA, stock concentration 0.5 M (Sigma Aldrich) PEG 8000 (Applichem) Ethanol (Thermo Fisher Scientific) GeneRuler 1 kb+ DNA ladder bands (Thermo Fisher Scientific) 20x reserve of TAE buffer (Thermo Fisher Scientific) Potassium chloride (Sigma Aldrich) Lithium chloride (Sigma Aldrich) Cesium chloride (Sigma Aldrich) Ammonium chloride (Sigma Aldrich) Ammonium sulfate (Thermo Fisher Scientific) Example 1 Effects of different concentrations of magnesium ions and nucleotide salts (dNTP salts) on DNA yield in rolling circle amplification (RCA) reactions (using lithium, sodium, potassium, cesium, and ammonium cations as counterions).

[0138] introduce

[0139] The concentration of magnesium ions in the reaction buffer is crucial for optimal DNA synthesis via DNA polymerase. Low magnesium ion concentrations have been reported to result in little or no DNA synthesis, while high concentrations often lead to nonspecific products and cause erroneous integration of dNTPs, resulting in increased replication errors. Because magnesium binds to the phosphate moiety of each dNTP, common practice is to use a magnesium ion concentration equal to or higher than that of the dNTPs used (Dean, FB, Nelson, JR, Giesler, TL, & Lasken, RS (2001). Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed RollingCircle Amplification. Genome Research, 11(6), 1095–1099. http: / / doi.org / 10.1101 / gr.180501). Magnesium-dNTPs are an absolute requirement for high-accuracy DNA synthesis via DNA polymerase. Magnesium also binds to DNA and can influence structural changes and form cross-links between individual strands at concentrations higher than those required for DNA synthesis.

[0140] To achieve industrially relevant enzymatic production of DNA, the concentration of dNTPs used in the reaction must be maximized to obtain the highest DNA yield. Furthermore, the reaction needs to be both efficient and accurate. Commercially available dNTPs are sodium or lithium salts, typically containing four monovalent metal cations per molecule. Most publications on DNA synthesis neglect the nature of counterions and their potential influence on magnesium-dNTP formation. Increasing the concentration of dNTPs in the reaction can increase the concentration of monovalent counterions by up to four times, thus they have a potential impact on the DNA amplification reaction.

[0141] Therefore, understanding the magnesium-monovalent magnesium counterion kinetics in DNA synthesis is crucial for maximizing DNA yield at the lowest possible magnesium concentration to achieve the highest accuracy of DNA products.

[0142] The following experiments evaluated the effects of increasing initial magnesium concentrations (5 mM, 10 mM, 20 mM, and 40 mM) and different dNTP salts on the yield of DNA amplified via RCA (rolling circle amplification).

[0143] Reaction setup

[0144] The reaction was set up on a 100 μl scale as follows: a denaturing mixture was prepared and placed at room temperature while the reaction mixture was assembled. These were then mixed, and DNA polymerase and pyrophosphatase were added. Table 1 shows the experimental protocol.

[0145] Before processing, incubate the RCA reaction at 30°C for at least 48 hours.

[0146] Table 1 - RCA Reaction Components

[0147] Sample processing procedures

[0148] After RCA for 48 hours, add 1.5 molar excess EDTA to MgCl2 and bring the reaction volume to 800 μl with water. Shake vigorously for 15 minutes and place on a vortex mixer until the reaction is completely mixed. Then, bring the reaction volume to 1 ml in 1 M NaCl by adding 200 μl of 5 M NaCl. Next, precipitate the polyp DNA by adding 100 μl of 50% (w / v) PEG 8000. Shake the mixture vigorously for 15 minutes to ensure complete precipitation, then centrifuge at 13,000 rpm for 10 minutes. Carefully discard the supernatant and wash the precipitate with 500 μl of 100% ethanol. Centrifuge the precipitate again at 13,000 rpm for 10 minutes, then carefully discard the ethanol supernatant. Dry the precipitate for 5 minutes to evaporate any residual ethanol, resuspend it in 1 ml of water, and incubate overnight on a vortex mixer.

[0149] The concentration of the reacting DNA was quantified based on UV absorbance measurements using an Implen NP80 nanophotometer. Data with a 10-fold increase in reaction volume were corrected, and concentrations are expressed as g / L of the original volume relative to the concentration of dNTPs used.

[0150] result

[0151] Tables 2 to 5 and Figure 1 and 2 This indicates that the initial concentration of magnesium and the initial concentration of different dNTP salts affect the yield of raw DNA. The values ​​in parentheses represent the magnesium / dNTP ratio at which each type of dNTP salt achieves the highest DNA yield.

[0152] Table 2 - MgCl₂ at a reaction concentration of 5 mM 2 Peak production is highlighted in bold; the numbers in parentheses are for magnesium per unit area. The proportion of dNTPs:

[0153] Table 3 - MgCl₂ at a reaction concentration of 10 mM 2 Peak production is highlighted in bold; the numbers in parentheses are for magnesium per unit area. The proportion of dNTPs:

[0154] Table 4 - MgCl₂ at a reaction concentration of 20 mM 2 Peak production is highlighted in bold; the numbers in parentheses are for magnesium per unit area. The proportion of dNTPs:

[0155] Table 5 - MgCl₂ at a reaction concentration of 40 mM 2 Peak production is highlighted in bold; the numbers in parentheses are for magnesium per unit area. The proportion of dNTPs:

[0156] Data in Tables 2 through 5 show that the highest DNA yields were obtained using non-commercially available potassium, ammonium, and cesium dNTP salts. By using these dNTP salts to counteract the ions and increasing the magnesium concentration to 40 mM, dNTPs with starting concentrations up to 50 mM can be used, and efficient conversion to DNA can be achieved via Phi29 DNA polymerase.

[0157] Lithium-dNTPs are poor substrates for DNA synthesis, requiring significantly higher levels of magnesium compared to other monovalent cations. In fact, peak DNA yields at 40 mM magnesium (4.328 g / L) are achieved with only 20 mM dNTPs. Sodium-dNTPs outperform their lithium equivalents, achieving peak DNA yields at 40 mM magnesium (6.897 g / L) with only 30 mM dNTPs.

[0158] Ammonium is the best counterion for dNTPs, achieving the highest DNA yield (13.44 g / L) at the highest initial concentration of dNTPs (50 mM dNTPs and 40 mM MgCl2) while maintaining a magnesium / dNTP ratio of 0.8. Data trends suggest that further increasing the concentration of MgCl2 should further enhance the initial concentration of ammonium-dNTPs and their integration into DNA.

[0159] Potassium-dNTPs also outperformed their lithium and sodium counterparts in terms of DNA yield and magnesium / dNTP ratio of 0.80 at high dNTP concentrations (50 mM and 40 mM MgCl2). Under the reaction conditions, the performance of potassium-dNTPs was almost comparable to that of ammonium-dNTPs.

[0160] In this method, at MgCl2 concentrations of 5 mM and 10 mM, the highest DNA yields (5.719 g / L and 8.262 g / L) were achieved with cesium-dNTPs at initial concentrations of 25 mM and 30 mM, respectively. The magnesium / dNTP ratio at 5 mM MgCl2 and 25 mM dNTPs was 0.2, the lowest among all the observed data. Therefore, the use of cesium-dNTPs is advantageous when it is beneficial to use the lowest possible concentration of magnesium ions while still producing high yields (beneficial to the outcome of the DNA amplification process).

[0161] Among the other monocations studied, the ammonium ion is unique because it is polyatomic and entirely nonmetallic. It acts as a pH buffer and exists as a 50% aqueous solution of ammonia (NH3) at a pKa of 9.24. The volatility of NH3 allows for the use of DNA processing techniques, such as low-pressure evaporation, that are impossible with metallic monocations.

[0162] Figure 1 The graph is a graphical representation of the data shown in Tables 2 to 5, and shows the obtained raw DNA yield (g / l) relative to the theoretical DNA yield (g / l) corresponding to the total initial / starting nucleotide salt concentration (mM) at different concentrations of magnesium chloride.

[0163] Figure 2 This graph shows the dNTP salt concentration (mM) for maximizing raw DNA yield relative to different magnesium ion concentrations. It clearly shows that lithium-dNTPs and sodium-dNTPs are most dependent on magnesium, but other counterions are less dependent on magnesium.

[0164] Table 6 includes the ionic radii of monovalent counterions at different coordination numbers. A clear relationship exists between the size of the counterion (relative to magnesium) and the magnesium concentration required to utilize high levels of dNTPs. Larger cations (e.g., potassium, cesium, and ammonium) are far superior to sodium, and especially far superior to lithium.

[0165] Table 6 - Atomic radii of counter ions:

[0166] Reference: http: / / abulafia.mt.ic.ac.uk / shannon / ptable.php, Shriver & Atkins

[0167] Therefore, DNA yield in industrial processes can be increased by selectively using dNTP salt counterions. This can be achieved by counterions affecting dNTPs, DNA, and the released phosphate (PO4) groups. 3-The differences in the affinity of anions and the competitive kinetics with divalent magnesium cations, as shown in Example 6, are used to mediate this process.

[0168] Example 2

[0169] Effects of different concentrations of magnesium ions and fixed concentrations of dNTP salts (using lithium, sodium, potassium, cesium, and ammonium cations as counterions) on rolling circle amplification (RCA) reactions and DNA yield.

[0170] Introduction & Reaction Setup

[0171] This experiment was designed to determine the minimum magnesium ion concentration required to integrate a fixed amount of dNTPs (10 mM) into the method at the outset. The RCA reaction and treatment were performed as described in Example 1. At the outset, the concentration of dNTPs (as lithium, sodium, potassium, cesium, and ammonium salts) was fixed at 10 mM in the method, and the reactions were carried out in the standard RCA buffer used in Example 1 with supplementation of 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM MgCl2.

[0172] result

[0173] The results show that, contrary to widely held views, an effective dNTP integration requires a magnesium / dNTP ratio of at least 1:1, and that when dNTPs with counterions different from lithium are used, magnesium levels in the RCA reaction can be reduced to levels far below this ratio while additionally increasing DNA yield.

[0174] from Figure 3 It can be seen that the yields of both sodium-dNTPs and lithium-dNTPs are strongly dependent on magnesium levels. Although the DNA production from potassium-dNTPs is slightly reduced at 2 mM MgCl2, this form of dNTP salt, as well as cesium-dNTPs and ammonium-dNTPs, shows lower dependence on magnesium ion concentration. This suggests that the general assumption of an optimal magnesium / dNTP ratio of 1:1, without considering the type of dNTP counterion, is misleading. The data indicate that this ratio can be reduced to 0.2:1 using alternative counterions such as lithium and sodium.

[0175] Example 3

[0176] Rolling circle amplification (RCA) reactions were performed in minimal buffer at fixed levels of magnesium ions and increasing concentrations of dNTP salts (with lithium, sodium, potassium, and ammonium cations as counterions); the effect on DNA yield.

[0177] Introduction & Reaction Setup

[0178] To eliminate the potential counterion effects of buffer components, experiments were then conducted in a minimal buffer consisting only of 30 mM Tris HCl (pH 7.9) supplemented with 5 mM MgCl2. These reactions investigated the effect of increasing initial dNTP salt concentrations (from 2.5 mM to 20 mM dNTPs, provided as lithium, sodium, potassium, or ammonium salts) in reactions containing 5 mM MgCl2.

[0179] Table 7 - RCA reaction components with minimal buffer solution

[0180] DNA processing and quantification were performed as described in Example 1.

[0181] The results are as follows Figure 4 As shown.

[0182] Data showed that RCA was performed in the absence of 30 mM KCl and 5 mM (NH4)2SO4 in the standard reaction buffer. The observed trend of increasing yield relative to the concentration of dNTP salts with variable counterions was consistent with the data given in Example 1, confirming that ammonium-dNTPs outperformed other counterion dNTP salts.

[0183] Figure 4 It has been confirmed that altering the dNTP salt counterion enables RCA to proceed at higher dNTP concentrations and correspondingly increases yield.

[0184] Table 8 - At 5 mM MgCl 2 The original DNA production of various counterion dNTPs under minimal buffer conditions Quantity. Peak production is highlighted in bold, and the magnesium / dNTP ratio is shown in parentheses:

[0185] Example 4

[0186] Rolling circle amplification (RCA) reactions at different concentrations of magnesium ions and ammonium-dNTPs were used to determine the highest original DNA yield.

[0187] Introduction & Reaction Setup

[0188] These reactions are designed to extend Example 3 ( Figure 4 The experimental data shown in the figure were used to find the limits of DNA yield by increasing the concentration of ammonium-dNTPs at different magnesium concentrations. Basically, as described in Example 3, the RCA reaction and DNA treatment were performed in a minimal buffer solution.

[0189] result

[0190] Table 9 - Different MgCl₂ values 2 The original DNA yield of ammonium-dNTPs at the lowest possible concentration in a minimal buffer. Peak Production figures are highlighted in bold, and the numbers in parentheses represent the magnesium / dNTP ratio:

[0191] Data indicate that by using ammonium counterion dNTPs, the initial concentration of dNTPs in the reaction can be further increased (up to 80 mM), resulting in very high levels of DNA. This was achieved by significantly increasing the concentration of MgCl2 to 80 mM in the minimum buffer. Even with 80 mM MgCl2 and 80 mM ammonium-dNTPs, it was clear that peak DNA yield was not reached. Adding more dNTPs should further increase DNA yield. Under conditions where the expected magnesium / dNTP ratio will be <1, increasing the concentrations of MgCl2 and ammonium-dNTPs (above 80 mM) should produce even higher levels of DNA.

[0192] Example 5

[0193] Determine the productivity limit of RCA in a water-magnesium chloride mixture.

[0194] Introduction & Reaction Setup

[0195] DNA amplification experiments were then conducted in reaction medium without Tris buffer or other salts conventionally essential for optimal DNA amplification, using 10 mM, 20 mM, and 40 mM MgCl2 and a range of potassium-, cesium-, and ammonium-dNTPs. Lithium and sodium-dNTPs were omitted here because other cations were superior during screening. Besides magnesium and dNTP counterions, the only other cations in the reaction included 5 mM sodium ions from NaOH used for template denaturation. Experiments were conducted to determine whether the dNTPs themselves and the phosphate byproducts of the reaction could maintain the pH levels and physicochemical conditions required for efficient DNA initiation to promote Phi29 DNA polymerase activity.

[0196] result

[0197] Table 10 - RCA reaction components without Tris buffer

[0198] DNA processing and quantification were performed in accordance with the procedure described in Example 1.

[0199] Table 11 - Containing 10 mM MgCl 2 Buffer-free medium. Peak yields are highlighted in bold, in parentheses. The numbers represent the magnesium / dNTP ratio:

[0200] Table 12 - Containing 20 mM MgCl 2 Buffer-free medium. Peak yields are highlighted in bold, in parentheses. The numbers represent the magnesium / dNTP ratio:

[0201] Table 13 - Contains 40 mM MgCl 2 Buffer-free medium. Peak yields are highlighted in bold, in parentheses. The numbers represent the ratio of [Mg] to [dNTP]:

[0202] Experimental data show that reactions using potassium-dNTPs yielded variable results in the absence of Tris buffer. On the other hand, cesium-dNTPs and ammonium-dNTPs, using increasing concentrations of magnesium ions and dNTPs, yielded progressively higher DNA yields. Cesium-dNTPs performed significantly better under these unbuffered conditions compared to the standard buffer environment (see Table 5). High DNA yields were recorded at 40 mM MgCl2 and 50 mM cesium-dNTPs (magnesium / dNTP ratio of 0.80). There was no significant difference in DNA yield between buffered and unbuffered conditions using ammonium-dNTPs. High DNA yields were observed at 40 mM MgCl2 and 60 mM ammonium-dNTPs (magnesium / dNTP ratio of 0.67).

[0203] Example 6

[0204] Effects of other counterions on DNA amplification using ammonium-dNTPs

[0205] Introduction & Reaction Setup

[0206] This experiment was conducted to demonstrate the effects of lithium, sodium, and potassium cations on DNA yield obtained by using RCA with ammonium-dNTPs.

[0207] Table 14 - Reaction Components:

[0208] The reactions were established as shown in Table 14. Four sets of experiments were conducted, containing initial concentrations of 17.5 mM, 25 mM, 35 mM, and 50 mM ammonium-dNTPs, and 5 mM, 10 mM, 20 mM, and 40 mM MgCl2, respectively. LiCl, NaCl, KCl, or NH4Cl were added to each set at total concentrations of 70 mM, 100 mM, 140 mM, and 200 mM, respectively. This resulted in additional cation concentrations competing with the ammonium dNTP counterion concentration. Furthermore, the ammonium concentration doubled with the addition of NH4Cl. The magnesium / dNTP ratio was less than 1.0 in each set of experiments.

[0209] DNA processing and quantification were performed essentially as described in Example 1.

[0210] The results are as follows Figure 5As shown.

[0211] Figure 5 The results showed that when using ammonium-dNTPs, cesium, ammonium, and potassium ions did not inhibit DNA synthesis. Furthermore, the ammonium concentration could even be doubled without affecting DNA yield.

[0212] In contrast, lithium and sodium have inhibitory effects, with lithium being more inhibitory than sodium. Therefore, the presence of lithium and sodium should be avoided in industrial DNA production processes, which require high concentrations of dNTPs to achieve high DNA yields.

[0213] Example 7

[0214] Studying the buffering effect of dNTPs on DNA synthesis reactions

[0215] Introduction & Reaction Setup

[0216] This experiment was conducted to observe the ability of dNTP salts to buffer the reaction mixture in the absence of any specific buffer.

[0217] Table 15 - In 10 mM MgCl₂ 2 Experimental setup for pH measurement

[0218] Table 16 - At 20 mM MgCl 2 Experimental setup for pH measurement

[0219] Table 17 At 30 mM MgCl 2 Experimental setup for pH measurement

[0220] Mix the reaction components according to the proportions shown in the table above to a final volume of 50 μl. Then measure the pH of the mixture using a Mettler Toledo SevenCompact™ S220 pH meter equipped with an InLab® Micro pH electrode.

[0221] Figure 6The pH values ​​measured for a range of dNTP concentrations (cesium and ammonium salts) in the presence of 10 mM, 20 mM, and 40 mM MgCl2 are shown. The NaOH concentration is used to denature the template DNA used in the DNA synthesis reactions. All other DNA synthesis reaction components were omitted for the purposes of this experiment, as they are known not to affect the initial pH. No specific pH-stabilizing buffer (e.g., Tris) was added in any case. At dNTP salt concentrations less than 30 mM, the greater buffering capacity of ammonium dNTPs relative to cesium dNTPs was evident and expected. Interestingly, at dNTP salt concentrations greater than 30 mM, the mean pH values ​​for cesium dNTP and ammonium dNTP reactions were similar, approximately 7 and 7.5, respectively. The data suggest that, when present at sufficient concentrations, the phosphate groups of the dNTPs themselves are used to adjust the pH to approximately 7. Since DNA polymerases can operate efficiently at pH around 7, this is an advantage for industrial-scale synthesis reactions that require high concentrations of dNTP salts. Importantly, it demonstrates that for industrial-scale reactions, DNA synthesis can be carried out with no or low concentrations of specific buffer solutions to achieve high productivity.

Claims

1. A cell-free process for enzymatic synthesis of DNA, comprising using a nucleotide salt, wherein the salt comprises a monovalent cation with an ionic radius greater than that of a sodium ion.

2. The cell-free process according to claim 1, wherein the nucleotide salt is present at a concentration greater than 10 mM.

3. A cell-free process for enzymatic synthesis of DNA, comprising using a nucleotide salt, wherein the nucleotide salt is present at a concentration of at least 10 mM and is: (a) Nucleotide salts containing monovalent cations with ionic radii greater than that of sodium ions, or (b) Two or more nucleotide salts, each containing a different monovalent cation, wherein at least one cation has an ionic radius greater than that of a sodium ion.

4. The cell-free process according to any of the preceding claims, wherein the nucleotide salt is present at a concentration of at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM, or at least 40 mM.

5. The cell-free process according to any of the preceding claims, wherein the one or more monovalent cations are independently selected from alkaline earth metals, transition metals or polyatomic ions.

6. The cell-free process according to claim 5, wherein the one or more monovalent cations are independently selected from the following list, including potassium, ammonium, ammonium derivatives, rubidium, cesium or francium.

7. The cell-free process according to any of the preceding claims, wherein the cell-free process further comprises using one or more primers or primases.

8. The cell-free process according to any of the preceding claims, wherein the cell-free process further comprises using a template.

9. A cell-free process for synthesizing DNA according to any of the preceding claims, wherein the cell-free process further comprises using one or more divalent metal cations, the divalent metal cations preferably selected from the list below, including magnesium, manganese, calcium, beryllium, zinc and strontium.

10. The cell-free process according to claim 9, wherein the ratio of the divalent metal cation to the nucleotide in the reaction mixture is equal to or less than 1:1, preferably less than 1:

1.

11. The cell-free process according to any of the preceding claims, wherein the process uses sodium nucleotides and / or lithium salts at a maximum concentration of 10 mM.

12. The cell-free process according to any of the preceding claims, wherein the process further comprises using a chemical denaturant, preferably sodium hydroxide, potassium hydroxide or ammonium hydroxide, and pyrophosphatase.

13. The cell-free process according to claim 12, wherein, No pH buffer is added to the process, and preferably, no additional salt or detergent is added.

14. The cell-free process according to claim 13, wherein the nucleotide salt comprises cesium ions.

15. The cell-free process according to claim 12, wherein a pH buffer is added, but no additional salt or detergent is added.

16. The cell-free process according to claim 15, wherein the nucleotide salt comprises ammonium ions.

17. The cell-free process according to any of the preceding claims, wherein the process is used for large-scale DNA synthesis, preferably at least 3 g / L.

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

19. The use according to claim 18, wherein the enzymatic cell-free synthesis of DNA is carried out in the presence of low levels of divalent cations, optionally, the ratio of divalent cations to nucleotides is 0.2:1 to 0.8:1, preferably 0.2:1 to 0.5:

1.

20. The use according to claim 18, wherein cell-free DNA synthesis is carried out in a minimal buffer, said buffer optionally containing only a pH buffer and free of detergents or additional salts.

21. A method for amplifying a DNA template using a DNA polymerase, wherein the ratio of divalent cations to nucleotides in the reaction mixture is maintained at 0.5:1 or lower, the method comprising using a nucleotide salt containing cesium ions.

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

23. A cell-free method for amplifying a DNA template, the method comprising contacting the template and a DNA polymerase with nucleotides in the form of a salt, the amount of the nucleotides being equal to or greater than 40 mM, preferably greater than 60 mM or optionally greater than 80 mM, wherein the salt comprises ammonium ions.

24. An enzymatic DNA synthesis carried out under conditions of reduced concentration of divalent cations, preferably magnesium, comprising the use of nucleotides in the form of salts, said salts comprising monovalent cations with ionic radii greater than that of sodium ions.

25. The cell-free process, use, method, cell-free method or enzymatic synthesis according to any of the preceding claims, wherein the enzyme is a DNA polymerase, optionally a strand displacement polymerase.

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