DNA SYNTHESIS YIELD IMPROVEMENTS
20230091493 · 2023-03-23
Assignee
Inventors
Cpc classification
C12Q2527/125
CHEMISTRY; METALLURGY
C12P19/34
CHEMISTRY; METALLURGY
C12Y207/07007
CHEMISTRY; METALLURGY
C12Q2527/125
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to an improved process for synthesis of deoxyribonucleic acid (DNA), in particular cell-free enzymatic synthesis of DNA, preferably on a large or industrial scale, with an improved yield and/or with an improved efficiency. The invention requires the use of nucleotide complexes wherein the nucleotide is associated with a mixture of divalent and monovalent cations. Preferably, the divalent cation may be magnesium or manganese.
Claims
1. A cell-free process for the enzymatic synthesis of DNA in solution comprising obtaining a nucleotide complex, where said nucleotide complex comprises a nucleotide associated with between 0.2 and 2 divalent cations and between 0.2 and 2.5 monovalent cations per nucleotide and adding a nucleotidyltransferase.
2. A cell-free process according to claim 1 wherein the nucleotide complex is a salt.
3. A cell-free process according to any one of claims 1 or 2 wherein said nucleotide complex is electrically neutral.
4. A cell-free process according to claim 3 wherein in order to obtain electrical neutrality the complex associates with one or more hydrogen or hydronium ions.
5. A cell-free process according to any preceding claim wherein the complex is associated with between about 0.5 and 1.5 divalent cations, preferably 1 divalent cation per nucleotide entity.
6. A cell-free process according to any preceding claim wherein the complex is associated with between about 0.2 and 2 monovalent cations.
7. A cell-free process according to any preceding claim wherein said divalent cations are independently selected from: magnesium (Mg.sup.2+), beryllium (Be.sup.2+), calcium (Ca.sup.2), strontium (Sr.sup.2+), manganese (Mn.sup.2+) or zinc (Zn.sup.2+), preferably Mg.sup.2+ or Mn.sup.2+.
8. A cell-free process according to any preceding claim wherein said monovalent cations are independently selected from: an alkali metal, a transition metal, or a polyatomic ion.
9. A cell-free process according to claim 8 wherein said monovalent cations may be independently selected from a polyatomic ion such as an oxonium ion, preferably ammonium or a derivative thereof.
10. A cell-free process according to claim 8 wherein said monovalent cations may be an alkali metal, independently selected from: lithium (Li.sup.+), sodium (Na.sup.+), potassium (K.sup.+), rubidium (Rb.sup.+), caesium (Cs.sup.+) or francium (Fr.sup.+).
11. A cell-free process according to any preceding claim, wherein said nucleotide complex in solution is obtained by mixing nucleotides complexed with divalent cations and a solution of nucleotides complexed with monovalent cations, preferably wherein the divalent and monovalent cations are present at a ratio of less than 4:1 cation:nucleotide, optionally 3.5:1, 3:1 or 2.5:1 or below.
12. A cell-free process according to claim 11 wherein said nucleotide complex with divalent cations is poorly soluble until mixing with nucleotides complexed with monovalent cations.
13. A cell-free process according to claims 11 and 12 wherein the nucleotide complex is soluble.
14. A cell-free process according to any preceding claim wherein said nucleotide complex is obtained at a concentration of at least 30 mM.
15. A cell-free process according to any preceding claim wherein said nucleotide complex is obtained at a concentration of at least 40 mM.
16. A cell-free process according to any preceding claim wherein said nucleotide complex and nucleotidyltransferase form a reaction mixture.
17. A cell-free process according to claim 16 wherein further components are added to the reaction mixture, including but not limited to any one or more of the following: a) template nucleic acid; b) primer; c) primase; d) denaturing agent, such as sodium or ammonium hydroxide; e) buffering agents; including buffering salts; f) pyrophosphatase; and/or g) magnesium or manganese salts
18. A cell-free process according to claim 17 wherein magnesium or manganese salts are added to the reaction mixture as a co-factor for the nucleotidyltransferase, such that the total ratio of the magnesium and/or manganese to nucleotide does not exceed 2:1.
19. A cell-free process according to any preceding claim wherein said nucleotidyltransferase is a DNA polymerase, preferably a strand-displacing DNA polymerase.
20. A cell-free process according to claim 19 wherein said nucleotidyltransferase is capable of isothermal DNA synthesis.
21. A cell-free process according to any one of claims 1-18 wherein said nucleotidyltransferase does not require a template.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] The present invention will be described further below with reference to exemplary embodiments and the accompanying drawings, in which:
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DETAILED DESCRIPTION
[0071] The present invention relates to cell-free processes for large scale synthesis of DNA. The processes of the invention may allow for a high throughput synthesis of DNA.
[0072] The deoxyribonucleic acid (DNA) synthesised according to the present invention can be any DNA molecule. The DNA may be single stranded or double stranded. The DNA may be linear. The DNA may be processed to form circles, particularly minicircles, single stranded closed circles, double stranded closed circles, double stranded open circles, or closed linear double stranded DNA. The DNA may be allowed to form, or processed to form a particular secondary structure, such as, but not limited to hairpin loops (stem loops), imperfect hairpin loops, pseudoknots, or any one of the various types of double helix (A-DNA, B-DNA, or Z-DNA). The DNA may also form hairpins and aptamer structures.
[0073] The DNA synthesised may be of any suitable length. Lengths of up to or exceeding 77 kilobases may be possible using the processes of the invention. More particularly, the length of DNA which may be synthesised according to the processes of the invention may be in the order of up to 60 kilobases, or up to 50 kilobases, or up to 40 kilobases, or up to 30 kilobases. Preferably the DNA synthesised may be 100 bases to over 77 kilobases, 500 bases to 60 kilobases, 200 bases to 20 kilobases, more preferably 200 bases to 15 kilobases, most preferably 2 kilobases to 15 kilobases.
[0074] The amount of DNA synthesised according to the processes of the present invention may exceed 9.75 g/l. It is preferred that the amount of DNA synthesised 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 more. A preferred amount of DNA synthesised is 5 g/l. The amount of DNA produced may be described as industrial or commercial quantities, on a large-scale or mass production. The DNA produced by the processes of the invention may be uniform in quality, namely in DNA length and sequence. The processes may thus be suitable for large scale synthesis of DNA. The process may be uniform in terms of fidelity of synthesis.
[0075] Alternatively, the amount of DNA produced in the synthesis reaction may be compared to the theoretical maximum yield which would be achieved if 100% nucleotides were incorporated into the synthesised DNA. The methods of the invention not only improve the total yield obtained, but also the efficiency of the process, meaning that more of the supplied nucleotides are incorporated into the synthesised DNA product than in previous methods. Yields obtainable by the methods of the invention exceed 50% of the theoretical maximum, up to and exceeding 90% of the theoretical maximum. Therefore, the proportion of the theoretical maximum yield achieved by methods of the invention include 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% and 95% or greater. Conventionally, using commercially available nucleotide salts, yields achieved could be disappointing, due to effects of the ions that may be inhibitory to the process.
[0076] The DNA is synthesised in an enzymatic reaction. This enzymatic synthesis may involve the use of any DNA synthesising enzyme, a nucleotidyltransferase, capable of adding a nucleotide to a nascent polynucleotide chain, most notably a polymerase enzyme or a modified polymerase enzyme. These are discussed further below. The DNA synthesis may be de novo and not require a template. The enzymatic synthesis may also require the use of a template for the DNA synthesis. This template can be any suitable nucleic acid depending on the polymerase, but is preferably a DNA template.
[0077] The template may be any suitable template, merely providing the instructions for the synthesis of the DNA by including a particular sequence. The template may be single stranded (ss) or double stranded (ds). The template may be linear or circular. The template may include natural, artificial or modified bases or a mixture thereof.
[0078] The template may comprise any sequence, either naturally derived or artificial.
[0079] The template may be of any suitable length. Particularly, the template may be up to 60 kilobases, or up to 50 kilobases, or up to 40 kilobases, or up to 30 kilobases. Preferably the DNA template may be 10 bases to 100 bases, 100 bases to 60 kilobases, 200 bases to 20 kilobases, more preferably 200 bases to 15 kilobases, most preferably 2 kilobases to 15 kilobases.
[0080] The template may be provided in an amount sufficient for use in the process by any method known in the art. For example, the template may be produced by PCR.
[0081] The whole or a selected portion of the template may be amplified in the process.
[0082] The template may comprise a sequence for expression. The DNA may be for expression in a cell (i.e. a transfected cell in vitro or in vivo), or may be for expression in a cell free system (i.e. protein synthesis). The sequence for expression may be for therapeutic purposes, i.e. gene therapy or a DNA vaccine. The sequence for expression may be a gene, and said gene may encode a DNA vaccine, a therapeutic protein and the like. The sequence may comprise a sequence which is transcribed into an active RNA form, i.e. a small interfering RNA molecule (siRNA). The sequence may comprise a sequence which is transcribed into mRNA, most particularly an mRNA for producing a vaccine.
[0083] If required, the template may be contacted with at least one polymerase, as described below.
[0084] The enzymatic DNA synthesis reaction may require at least one DNA synthesis enzyme (a nucleotidyltransferase). Preferably, the DNA synthesis enzyme is a polymerase. Polymerases link together nucleotides 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, such that it synthesises polymers 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 which provides a proofreading function and one or more others which do not. DNA polymerases having different mechanisms may be used e.g. strand displacement type polymerases and DNA polymerases replicating DNA by other methods. A suitable example of a DNA polymerase that does not have strand displacement activity is T4 DNA polymerase. Template-independent polymerases may be used, such as terminal transferases.
[0085] Modified polymerases may also be used. These may have been engineered to modify their characteristics, such as to remove their dependency upon a template, to change their temperature dependency or to stabilise the enzyme for use in vitro.
[0086] A polymerase may be highly stable, such that its activity is not substantially reduced by prolonged incubation under process conditions. Therefore, the enzyme preferably has a long half-life under a range of process conditions including but not limited to temperature and pH. It is also preferred that a polymerase has one or more characteristics suitable for a manufacturing process. The polymerase preferably has high fidelity, for example through having proofreading activity. Furthermore, it is preferred that a polymerase displays one or more of: high processivity, high strand-displacement activity and a low K.sub.m for dNTPs and DNA. A polymerase may be capable of using circular and/or linear DNA as template. The polymerase may be capable of using dsDNA or ssDNA as a template. It is preferred that a polymerase does not display DNA exonuclease activity that is not related to its proofreading activity. Further, the polymerase may be capable of using an alternative nucleic acid as a template.
[0087] The skilled person can determine whether or not a given polymerase displays characteristics as defined above by comparison with the properties displayed by commercially available polymerases, e.g. Phi29 (New England Biolabs, Inc., Ipswich, Mass., US), Deep Vent® (New England Biolabs, Inc.), Bacillus stearothermophilus (Bst) DNA polymerase I (New England Biolabs, Inc.), 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.). Where a high processivity is referred to, this typically denotes the average number of nucleotides added by a polymerase enzyme per association/dissociation with the template, i.e. the length of nascent extension obtained from a single association event.
[0088] Strand displacement-type polymerases are preferred. Preferred strand displacement-type polymerases are Phi29, Deep Vent and Bst DNA polymerase I or variants of any thereof. “Strand displacement” describes the ability of a polymerase to displace complementary strands on encountering a region of double stranded DNA during synthesis. The template is thus amplified by displacing complementary strands and synthesising a new complementary strand. Thus, during strand displacement replication, a newly replicated strand will be displaced to make way for the polymerase to replicate a further complementary strand. The amplification reaction initiates when a primer or the 3′ free end of a single stranded template anneals to a complementary sequence on a template (both are priming events). When DNA synthesis proceeds and if it encounters a further primer or other strand annealed to the template, the polymerase displaces this and continues its strand elongation. The strand displacement may release single stranded DNA which can act as a template for more priming events. The priming of the newly released DNA may lead to hyper-branching, and a high yield of products. It should be understood that strand displacement amplification methods differ from PCR-based methods in that cycles of denaturation are not essential for efficient DNA amplification, as double-stranded DNA is not an obstacle to continued synthesis of new DNA strands. Strand displacement amplification may only require one initial round of heating, to denature the initial template if it is double stranded, to allow the primer to anneal to the primer binding site, if a primer is used. Following this, the amplification may be described as isothermal, since no further heating or cooling is required. In contrast, PCR methods require cycles of denaturation (i.e. elevating temperature to 94 degrees centigrade or above) during the amplification process to melt double-stranded DNA and provide new single-stranded templates. During strand displacement, the polymerase will displace strands of already synthesised DNA. Further, it will use newly synthesised DNA as a template, ensuring rapid amplification of DNA.
[0089] A strand displacement polymerase used in a process of the invention preferably has a processivity of at least 20 kb, more preferably, at least 30 kb, at least 50 kb, or at least 70 kb or greater. In one embodiment, the strand displacement DNA polymerase has a processivity that is comparable to, or greater than phi29 DNA polymerase.
[0090] Strand displacement replication is, therefore, preferred. During strand displacement replication, the template is amplified by displacing already replicated strands, which have been synthesised by the action of the polymerase, in turn displacing another strand, which can be the original complementary strand of a double stranded template, or a newly synthesised complementary strand, the latter synthesised by the action of a polymerase on an earlier primer annealed to the template. Thus, the amplification of the template may occur by displacement of replicated strands through strand displacement replication of another strand. This process may be described as strand displacement amplification or strand displacement replication.
[0091] A preferred strand displacement replication process is Loop-mediated isothermal amplification, or LAMP. LAMP generally uses 4-6 primers recognizing 6-8 distinct regions of the template DNA. In brief, a strand-displacing DNA polymerase initiates synthesis and 2 of the primers form loop structures to facilitate subsequent rounds of amplification. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem-loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem-loop DNA and a new stem-loop DNA with a stem twice as long. Modified LAMP procedures can also be adopted, where fewer internal primers are required.
[0092] A preferred strand displacement replication process is rolling circle amplification (RCA). The term RCA describes the ability of RCA-type polymerases to continuously progress around a circular DNA template strand whilst extending a hybridised primer. The “primer” may be added, created by a primase or generated by nicking one strand of a double stranded template. This amplification leads to formation of linear single stranded products with multiple repeats of amplified DNA. The sequence of the circular template (a single unit) is multiply repeated within a linear product. For a circular template, the initial product of strand displacement amplification is a single stranded concatamer, which is either sense or antisense, depending on the polarity of the template. These linear single stranded products serve as the basis for multiple hybridisation, primer extension and strand displacement events, resulting in formation of concatameric double stranded DNA products, again comprising multiple repeats of amplified DNA. There are thus multiple copies of each amplified “single unit” DNA in the concatameric double stranded DNA products. RCA polymerases are particularly preferred for use in the processes of the present invention. The products of RCA-type strand displacement replication processes may require processing to release single unit DNAs. This is desirable if single units of DNA are required. Typical strand displacement conditions using Phi29 DNA polymerase include high levels of magnesium ions, for example 10 mM magnesium (normally as a chloride salt) in combination with 0.2 to 4 mM nucleotides (when presented as typical lithium or sodium salts).
[0093] In order to allow for amplification, according to some aspects one or more primers may also be required by the enzymatic DNA synthesis. If no template is used, the primers allow for a starting point for DNA synthesis and are designed to begin the synthesis reaction. If a template is used, the primers may be non-specific (i.e. random in sequence) or may be specific for one or more sequences comprised within the template. Alternatively, a primase enzyme may be supplied to generate the primer de novo. If the primers are of random sequence they allow for non-specific initiation at any site on the template. This allows for high efficiency of amplification through multiple initiation reactions from each template strand. Examples of random primers are hexamers, heptamers, octamers, nonamers, decamers or sequences greater in length, for example of 12, 15, 18, 20 or 30 nucleotides in length. A random primer may be of 6 to 30, 8 to 30 or 12 to 30 nucleotides in length. Random primers are typically provided as a mix of oligonucleotides which are representative of all potential combinations of e.g. hexamers, heptamers, octamers or nonamers in the template.
[0094] In one embodiment, the primers or one or more of the primers are specific. This means they have a sequence which is complementary to a sequence in the template from which initiation of amplification is desired. In this embodiment, a pair of primers may be used to specifically amplify a portion of the DNA template which is internal to the two primer binding sites. Alternatively, a single specific primer may be used. A set of primers may be employed.
[0095] Primers may be any nucleic acid composition. Primers may be unlabelled, or may comprise one or more labels, for example radionuclides or fluorescent dyes. Primers may also comprise chemically modified nucleotides. For example, the primer may be capped in order to prevent initiation of DNA synthesis until the cap is removed, i.e., by chemical or physical means. Primer lengths/sequences may typically be selected based on temperature considerations i.e. as being able to bind to the template at the temperature used in the amplification step. Primers may be RNA primers, such as those synthesized by a primase.
[0096] In certain aspects, the contacting of the template with the synthesis enzyme and one or more primers may take place under conditions promoting annealing of primers to the template. The conditions include the presence of single-stranded nucleic acid allowing for hybridisation of the primers. The conditions conventionally also include a temperature and buffer allowing for annealing of the primer to the template. Appropriate annealing/hybridisation conditions may be selected depending on the nature of the primer. An example of conventional annealing conditions, which may be used in the present invention include a buffer comprising 30 mM Tris-HCl pH 7.5, 20 mM KCl, 8 mM MgCl.sub.2. In the Examples, the reactions with the nucleotide complexes of the invention are performed in 30 mM Tris pH 8.0 as a buffering agent alone. However, the present inventors have described conditions herein with reduced buffer and divalent metal ion components that still allow for primer binding and these are discussed further below. The annealing may be carried out following denaturation using heat followed by gradual cooling to the desired reaction temperature.
[0097] However, amplification using strand displacement replication can also take place without a primer, and thus requires no hybridisation and primer extension to occur. Instead, the single stranded template self-primes by forming hairpins, which have a free 3′ end available for extension. The remaining steps of the amplification remain the same. Alternatively, a double stranded template can be nicked to allow for strand displacement replication to use one strand of the template itself as a primer. Those skilled in the art are aware of all methods for providing initiation of amplification from a template.
[0098] The template and/or polymerase are also contacted with nucleotides, as nucleotide complexes as defined herein. The combination of template, nucleotidyltransferase and nucleotide complexes may be described as forming a reaction mixture. The reaction mixture may also comprise one or more primers or a primase. The reaction mixture may independently also include one or more divalent metal cations, should sufficient not be supplied with the nucleotide complexes. The reaction mixture may further comprise a chemical denaturant. Such denaturants can be potassium, ammonium or sodium hydroxide. The reaction mixture may further comprise additional enzymes, such as a helicase or a pyrophosphatase. The reaction mixture may contain pH buffering agents, and in some aspects, it contains no additionally added pH buffering agents.
[0099] A nucleotide is a monomer, or single unit, of nucleic acids, and nucleotides are composed of a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group. Any suitable nucleotide may be used.
[0100] The nucleotides are present as complexes, and are thus associated with a mixture of divalent and monovalent cations. Monovalent cations are ionic species with a single positive charge, and may be a metal ion or a polyatomic ion, such as an oxonium ion. Divalent cations are ionic species with a double positive charge, and may be a metal ion or a polyatomic ion.
[0101] A counter-ion is the ion that accompanies or is associated with an ionic species (the nucleotide in the present invention) in order to partially or completely balance the charge on the ionic species.
[0102] A complex is generally understood to be a molecular entity formed by loose association involving two or more component molecular entities (ionic or uncharged), or the corresponding chemical species. A complex may be either an ion or an electrically neutral molecule, formed by the union of simpler substances (as compounds or ions) and held together by forces that are chemical (i.e., dependent on specific properties of particular atomic structures) rather than physical. The bonding between the components is normally weaker than in a covalent bond.
[0103] The nucleotide complexes may include monovalent metal ions, including but not limited to alkali metals (group 1): lithium (Li.sup.+), sodium (Na.sup.+), potassium (K.sup.+), rubidium (Rb.sup.+), caesium (Cs.sup.+) or francium (Fr.sup.+). Alternatively or additionally, the monovalent metal ion may be a transition metal (Group 11): copper (Cu.sup.+), silver (Ag.sup.+), gold (Au.sup.+) or roentgenium (Rg.sup.+). The alkali metals are preferred, and thus the preferred counter-ion may be lithium (Li.sup.+), sodium (Na.sup.+), potassium (K.sup.+), rubidium (Rb.sup.+), caesium (Cs.sup.+) or francium (Fr.sup.+).
[0104] The nucleotide complexes may include polyatomic monovalent ions. A polyatomic ion is an ion that contains more than one atom. This differentiates polyatomic ions from monatomic ions, which contain only one atom. Exemplary monovalent polyatomic cations include oxonium ions. An oxonium ion is any oxygen cation with three bonds. The simplest oxonium ion is the hydronium ion H.sub.3O. Other notable oxonium ions include ammonium (NH.sub.4.sup.+) and ionic derivatives of ammonium.
[0105] Derivatives of ammonium are also encompassed, and a non-limiting, exemplary list of these includes: monoalkyl ammonium, dialkyl ammonium, trialkyl ammonium, choline, quaternary ammonium and imidazolium. Those skilled in the art will be aware of further derivatives of ammonium that carry a single positive charge that are appropriate to use in the present invention.
[0106] The nucleotide complexes may include divalent cations. The divalent cations associated with the nucleotide in the complex may comprise one or more metals selected from the list consisting of: Mg.sup.2+, Be.sup.2+, Ca.sup.2+, Sr.sup.2+, Mn.sup.2+ or Zn.sup.2+, preferably Mg.sup.2+ or Mn.sup.2+. The ratio between the divalent metal cations and the nucleotide (nucleotide ion or nucleotide ionic species) may be about 1:1 in solution, but is preferably between 0.2:1 and 2:1, optionally 0.5:1 to 1.5:1. Ratios lower than 1:1 are desirable and are preferable in DNA synthesis since ratios higher than 1:1 may lead to some infidelity in DNA synthesis. The provision of the divalent cations in relation to the nucleotide complex may therefore reduce or remove the need to add additional divalent cations to the reaction mixture. However, should further be required, these divalent cations may be provided to the enzymatic DNA synthesis in the form of any suitable salt.
[0107] Between 0.2 and 2 divalent cations may be associated with the nucleotide complex. This range includes 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2 divalent cations per nucleotide complex. Those skilled in the art will appreciate the non-whole numbers represent a sharing of the divalent ion between nucleotide free acids.
[0108] The nitrogenous base may be adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). The nitrogenous base may also be modified bases, such as 5-methylcytosine (m5C), pseudouridine (Ψ), dihydrouridine (D), inosine (I), and 7-methylguanosine (m7G). The nitrogenous base may further be an artificial base. The concentration of nucleotide complexes may include any combination of the various nitrogenous bases.
[0109] It is preferred that the five-carbon sugar is a deoxyribose, such that the nucleotide is a deoxynucleotide.
[0110] The nucleotides may be in the form of deoxynucleoside triphosphate, denoted dNTP. This is a preferred embodiment of the present invention. Suitable dNTPs may include dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dTTP (deoxythymidine triphosphate), dUTP (deoxyuridine triphosphate), dCTP (deoxycytidine triphosphate), dITP (deoxyinosine triphosphate), dXTP (deoxyxanthosine triphosphate), and derivatives and modified versions thereof. It is preferred that the dNTPs comprise one or more of dATP, dGTP, dTTP or dCTP, or modified versions or derivatives thereof. It is preferred to use a mixture of dATP, dGTP, dTTP and dCTP or modified version thereof. Any suitable ratios of these dNTPs can be used, according to the needs of the reaction.
[0111] The nucleotide complexes, may already be in solution prior to mixing with the nucleotidyltransferase, or may need to be supplied as a solid for example as a powder, and dispersed in solution. The nucleotide complexes may comprise modified nucleotides. The nucleotide complexes may be provided in a mixture of one or more suitable bases, preferably, one or more of adenine (A), guanine (G), thymine (T), cytosine (C). Two, three or preferably all four nucleotides (A, G, T, and C) are used in the process to synthesise DNA. These nucleotide complexes may all be present in substantially equal amounts or more of one or two may be provided, depending on the nature of the DNA to be synthesised.
[0112] The nucleotides may all be natural nucleotides (i.e. unmodified), they may be modified nucleotides that act like natural nucleotides and are biologically active (i.e. LNA nucleotides—locked nucleic acid), they may be modified and biologically inactive or they may be a mixture of unmodified and modified nucleotides, and/or a mixture of biologically active and biologically inactive nucleotides. Each type (i.e. base) of nucleotide may 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 appropriate complexes.
[0113] In one aspect of the invention, the nucleotides or nucleotide complexes are present at a concentration of at least 30 mM. According to this aspect, the nucleotides or nucleotide salts may be present in the reaction mixture at a concentration of more than more than 30 mM, more than 35 mM, more than 40 mM, more than 45 mM, more than 50 mM, more than 55 mM, more than 60 mM, more than 65 mM, more than 70 mM, more than 75 mM, more than 80 mM, more than 85 mM, more than 90 mM, more than 95 mM, or more than 100 mM. Such concentrations are given as the concentration of nucleotide complex at the initiation or start of the process. The concentration is given after the addition of the nucleotide/nucleotide complexes, wherein the addition may be to the reaction mixture. The nucleotide complex may be any appropriate mixture of nucleotide complexes, with varying nitrogenous bases. The concentration applies to the sum total of nucleotide complexes present in at the start of the process, whatever their composition. Thus, for example, a 30 mM concentration of nucleotide salts may be any mixture of dCTP, dATP, dGTP and dTTP counter-ioned with appropriate monovalent and divalent cations.
[0114] It will be understood that nucleotides supplied as complexes may dissociate in water and other solvents to form an anionic nucleotide entity (nucleotide ion, nucleotide ionic species) and the associated cations.
[0115] It is a preferred part of any aspect of the present invention that the nucleotide complex is formed by a mixture of counter-ions The nucleotide complexes used in the processes of the inventions include a mixture of different cation species; notably at least one species of divalent cation and at least one species of monovalent cation. Preferably the ratio of monovalent cation:nucleotide is between 0.2:1 and 2.5:1, optionally between 0.5:1 to 2:1. Preferably the ratio of divalent cation:nucleotide is between 0.2:1 and 2:1, optionally between 0.5:1 to 1.5:1, preferably 0.5:1 up to 1:1. The enzymatic DNA synthesis may be maintained under conditions promoting synthesis of DNA, and this will depend upon the particular method selected.
[0116] Amplification of a template via strand displacement is preferred. Preferably, the conditions promote amplification of said template by displacement of replicated strands through strand displacement replication of another strand. The conditions comprise use of any temperature allowing for amplification of DNA, commonly in the range of 20 to 90 degrees centigrade. A preferred temperature range may be about 20 to about 40 or about 25 to about 35 degrees centigrade. A preferred temperature for LAMP amplification is about 50 to about 70 degrees centigrade.
[0117] Typically, an appropriate temperature for enzymatic DNA synthesis is selected based on the temperature at which a specific polymerase has optimal activity. This information is commonly available and forms part of the general knowledge of the skilled person. For example, where phi29 DNA polymerase is used, a suitable temperature range would be about 25 to about 35 degrees centigrade, preferably about 30 degrees centigrade. However, a thermostable phi29 may operate at a higher constant temperature. The skilled person would routinely be able to identify a suitable temperature for efficient amplification according to the processes of the invention. For example, a process could be carried out at a range of temperatures, and yields of amplified DNA could be monitored to identify an optimal temperature range for a given polymerase. The amplification may be carried out at a constant temperature, and it is preferred that the process is isothermal. Since strand displacement amplification is preferred there is no requirement to alter the temperature to separate DNA strands. Thus, the process may be an isothermal process.
[0118] Other conditions promoting DNA synthesis are conventionally thought to comprise the presence of suitable buffering agents/pH and other factors which are required for enzyme performance or stability. Suitable conventional conditions include any conditions used to provide for activity of polymerase enzymes known in the art.
[0119] For example, the pH of the reaction mixture may be within the range of 3 to 10, preferably 5 to 8 or about 7, such as about 7.5. pH may be maintained in this range by use of one or more buffering agents (also called pH buffering agents). The function of a buffering agent is to prevent a change in pH. Such buffers (buffering agents) include, but are not restricted to MES, Bis-Tris, ADA, ACES, PIPES, MOBS, MOPS, MOPSO, Bis-Tris Propane, BES, TES, HEPES, DIPSO, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS, phosphate, citric acid-sodium hydrogen phosphate, citric acid-sodium citrate, sodium acetate-acetic acid, imidazole and sodium carbonate-sodium bicarbonate. Preferred are buffering agents that do not provide further cations to the reaction mixture, nor complex with metal cations present in the reaction mixture as discussed previously.
[0120] A buffer is generally defined by a mixture of reaction components. Usually included is a buffering agent to maintain a stable pH; one or more additional salts composed of a cationic and anionic species i.e. sodium chloride, potassium chloride; and/or detergents such as Triton-X-100 to ensure optimal activity or stability of the enzymes. A minimal buffer is composed of only a buffering reagent with no additional salts or detergents provided, with the proviso that small amounts of cationic species may be present for DNA synthesis in which chemical denaturation is required. Surprisingly, using higher concentrations of nucleotide salts in the processes of the invention permits the use of these minimal buffers.
[0121] A “no buffer” system lacks a provided or defined pH buffering agent in the mixture of reaction components and lacks additional salts or detergents. This “no added buffering agent” system contains only the reaction components required for the DNA synthesis alone, and contains cationic species provided for chemical denaturation only (if required). Thus, in this system, there are no additional ions added beyond those that serve a specific purpose in the DNA synthesis reaction. The counter-ions provided with the nucleotides (as a complex) serve to stabilise the nucleotide prior to use in the process.
[0122] While the application of heat (exposure to 95° C. for several minutes) is used to denature double stranded DNA other approaches may be used which are more suitable for DNA synthesis. Double stranded DNA can be readily denatured by exposure to a high or low pH environment or where cations are absent or present in very low concentrations, such as in deionized water. The polymerase requires the binding of a short oligonucleotide primer sequence to a single stranded region of the DNA template to initiate its replication. The stability of this interaction and therefore the efficiency of DNA synthesis may particularly be influenced by the concentration of metal cations and particularly divalent cations such as magnesium (Mg.sup.2+) ions which may be seen as an integral part of the process.
[0123] The enzymatic DNA synthesis may also require the presence of additional divalent metal ions, namely divalent cations that are supplied externally to the nucleotide complex. The process may comprise the use of salts of divalent metal ions: magnesium (Mg.sup.2+), manganese (Mn.sup.2+), calcium (Ca.sup.2+), beryllium (Be.sup.2+), zinc (Zn.sup.2+) and strontium (Sr.sup.2+). The most often used divalent ions in DNA synthesis is magnesium or manganese, since these act as a cofactor in DNA synthesis. Any suitable anion may be utilised in such salts, whilst being mindful that the choice of anion can have an effect on the pH of the reaction mixture, and should be suitably accounted for.
[0124] Detergents may also be included in the reaction mixture in certain aspects. Examples of suitable detergents include Triton X-100™, Tween 20™ and derivatives of either thereof. Stabilising agents may also be included in the reaction mixture. Any suitable stabilising agent may be used, in particular, bovine serum albumin (BSA) and other stabilising proteins. Reaction conditions may also be improved by adding agents that relax DNA and make template denaturation easier. Such agents include, for example, dimethyl sulphoxide (DMSO), formamide, glycerol and betaine. DNA condensing agents may also be included in the reaction mixture. Such agents include, for example, polyethylene glycol or cationic lipid or cationic polymers.
[0125] However, in certain embodiments, these components may be reduced or removed from the reaction mixture, for example in the minimal or no added buffering agent systems.
[0126] It should be understood that the skilled person is able to modify and optimise synthesis conditions for the processes of the invention using these additional components and conditions on the basis of their general knowledge. Likewise the specific concentrations of particular agents may be selected on the basis of previous examples in the art and further optimised on the basis of general knowledge.
[0127] As an example, a suitable reaction buffer used in RCA-based methods in the art is 50 mM Tris HCl, pH 7.5, 10 mM MgCl.sub.2, 20 mM (NH.sub.4).sub.2SO.sub.4, 5% glycerol, 0.2 mM BSA, 1 mM dNTPs. A preferred reaction buffer used in the RCA amplification is usually 30 mM Tris-HCl pH 7.9, 30 mM KCl, 7.5 mM MgCl.sub.2, 10 mM (NH.sub.4).sub.2SO.sub.4, 4 mM DTT, 2 mM dNTPs. This buffer is particularly suitable for use with Phi29 DNA polymerase when conventional nucleotides are purchased.
[0128] A suitable reaction buffer for use with the nucleotide complexes of the invention is 60 mM Tris pH 8.0. A further suitable reaction buffer is 30 mM Tris pH 8.0. Alternative conditions include 30 mM Tris HCl, pH 7.9, 5 mM (NH.sub.4).sub.2SO.sub.4, and 30 mM KCl. Under certain circumstances, the enzymatic DNA synthesis may be conducted in water (“no added buffering agent”).
[0129] The enzymatic DNA synthesis may also comprise the use of one or more additional proteins. The template may be amplified in the presence of at least one pyrophosphatase, such as Yeast Inorganic pyrophosphatase. Two, three, four, five or more different pyrophosphatases may be used. These enzymes are able to degrade pyrophosphate generated by the polymerase from dNTPs during strand replication. Build-up of pyrophosphate in the reaction can cause inhibition of DNA polymerases and reduce speed and efficiency of DNA amplification. Pyrophosphatases can break down pyrophosphate into non-inhibitory phosphate. An example of a suitable pyrophosphatase for use in the processes of the present invention is Saccharomyces cerevisiae pyrophosphatase, available commercially from New England Biolabs, Inc.
[0130] Any single-stranded binding protein (SSBP) may be used in the processes of the invention, to stabilise single-stranded DNA. SSBPs are essential components of living cells and participate in all processes that involve ssDNA, such as DNA replication, repair and recombination. In these processes, SSBPs bind to transiently formed ssDNA and may help stabilise ssDNA structure. An example of a suitable SSBP for use in the processes of the present invention is T4 gene 32 protein, available commercially from New England Biolabs, Inc.
[0131] The yield of the reaction relates to the amount of DNA synthesised. The expected yield from a process according to the present invention may exceed 3 g/l. It is preferred that the amount of DNA synthesised 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 more. A preferred amount of DNA synthesised is 5 g/l. 30 mM nucleotide complex is capable of generating 9.74 g/l DNA. The present invention improves the yield possible from enzymatic synthesis of DNA. It is an object of the present invention to improve the yield of a cell-free enzymatic DNA synthesis process, such that DNA can be synthesised on a large scale in a cost-effective way. The present invention allows the manufacture/synthesis of DNA economically on an industrial scale using an enzymatic process catalysed by a DNA synthesis enzyme or polymerase. The present process allows the efficient incorporation of nucleotides into the DNA product. It is thought that the processes of the invention will allow reaction mixtures to be scaled up into several litres, including tens of litres. The improved yield, productivity or processivity may be compared to an identical reaction mixture where all of the nucleotides are supplied as conventional salts with monovalent cation counter-ions (generally lithium or sodium).
[0132] In one embodiment, the present invention relates to a process for enhancing the synthesis of DNA. This enhancement may be compared to an identical reaction mixture, with the exception that all of the nucleotides complexes used are exclusively monovalent cation counter-ions, or a mixture thereof.
[0133] In one aspect, the invention provides a cell-free process for synthesising DNA comprising contacting a DNA template with at least one nucleotidyltransferase in the presence of one or more nucleotide complexes, wherein each of said nucleotide complexes are associated with between 0.2 and 2 divalent cations and between 0.2 and 2.5 monovalent cations.
[0134] It is preferred that the concentration of nucleotides referred to herein is the starting concentration of nucleotides at the start of the process, the initial concentration when the reaction mixture is formed.
[0135] The invention may also relate to a cell-free process for synthesising DNA comprising contacting a DNA template with at least one nucleotidyltransferase in the presence of one or more nucleotide complexes in a concentration of over 30 mM. The invention provides a cell-free process for the enzymatic synthesis of DNA comprising the use of nucleotides supplied as complexes, wherein said each of said complexes are a nucleotide associated with between 0.2 and 2 divalent cations and between 0.2 and 2.5 monovalent cations, preferably wherein the nucleotide complexes are obtained, supplied or are present in a concentration greater than 30 mM.
[0136] The invention further provides an enzymatic DNA synthesis which is performed under conditions of reduced or even no further additionally supplied divalent cations, preferably magnesium, comprising the use of nucleotide complexes, wherein each of said complexes comprise a nucleotide associated with between 0.2 and 2 divalent cations and between 0.2 and 2.5 monovalent cations. The provision of the divalent cation in the nucleotide complex avoids the further use of divalent cation salts in the process. However, in certain circumstances, the amount of divalent cation salts such as magnesium, are reduced using the complexes of the invention.
The invention will now be described with reference to several non-limiting examples.
Example 1
[0137] The Effects of Mono-Cation Concentration in Nucleotide Complexes on DNA Synthesis
[0138] Materials and Methods
[0139] Reagents
[0140] The following reagents supplied were used in the presented examples: [0141] Solution 1-100 mM dATP:4Na.sup.+ [0142] Solution 2-100 mM dCTP:4Na.sup.+ [0143] Solution 3-100 mM dGTP:4Na.sup.+ [0144] Solution 4-100 mM dTTP: 4Na.sup.+ [0145] Solution 5-100 mM dATP: 4NH.sub.4.sup.+ [0146] Solution 6-100 mM dCTP: 4NH.sub.4.sup.+ [0147] Solution 7-100 mM dGTP: 4NH.sub.4.sup.+ [0148] Solution 8-100 mM dTTP: 4 NH.sub.4.sup.+ [0149] Solution 9-25 mM dATP:1.6 Mg.sup.2+ [0150] Solution 10-61 mM dCTP:2.0 Mg.sup.2+ [0151] Solution 11-34 mM dGTP: 1.8 Mg.sup.2+ [0152] Solution 12-91 mM dTTP: 2.5 Mg.sup.2+ [0153] Phi29 DNA polymerase, stock concentration 5.6 g/L (produced in-house) [0154] Thermostable pyrophosphatase, stock concentration 2000 U/ml (Enzymatics) [0155] DNA primer, stock concentration 5 mM (Oligofactory) [0156] Plasmid template: ProTLx-K B5X4 LUX 15-0-15-10-15 AT-STEM, stock concentration 0.832 g/L (produced in house) [0157] Nuclease free water (Sigma Aldrich) [0158] 1M NaOH (Sigma Aldrich) [0159] Magnesium Acetate, stock concentration 1M (Sigma Aldrich) [0160] Tris-base (Thermo Fisher Scientific) [0161] Tris-HCl (Sigma Aldrich) [0162] NaCl (Sigma Aldrich) [0163] EDTA, stock concentration 0.5 M (Sigma Aldrich) [0164] PEG 8000 (AppliChem)
[0165] Preparation of dNTP Mixes
[0166] For both the sodium complex (dNTP:4Na.sup.+) and ammonium complex (dNTP: 4NH.sub.4.sup.+) the individual dNTPS (solutions 1, 2, 3, 4 and 5, 6, 7, 8 respectively) were mixed 1:1:1:1 to produce a stock concentration of 100 mM dNTP mixture. The dNTP mixes were stored at −20° C. until ready for use.
[0167] For sodium/magnesium complexes (dNTPs:Na.sup.+/Mg.sup.2+) and ammonium/magnesium complexes (dNTPs:NH.sub.4.sup.+/Mg.sup.2+) dNTPS were mixed as follows to provide an equal molar amount of each mononucleotide
TABLE-US-00001 TABLE 1 Table 1: Volumes of dNTPS used to form monovalent:divalent dNTP mixes Magnesium Ammonium or Sodium dNTP complex dNTP complex Vol- Vol- [dXTP] ume [dXTP] ume dNTP mM (μl) dNTP mM (μl) dATP (soln 9) 25 1820 dATP (soln 1 or 5) 100 455 dCTP (soln 10) 61 745 dCTP (soln 2 or 6) 100 455 dGTP (soln 11) 34 1338.2 dGTP (soln 3 or 7) 100 455 dTTP (soln 12) 91 500 dTTP (soln 4 or 8) 100 455
[0168] Mixing the solutions in Table 1 in the volumes indicated yields a stock concentration of 58 mM dNTPs in a ratio of 1:1 with Mg.sup.2+ and 1:2 with either Na.sup.+ or NH.sub.4.sup.+. To achieve a final stock concentration of 100 mM dNTP mix, the final volume of 6223 μl was reduced to 3640 μl using an Eppendorf SpeedVac Concentrator Plus operating at a temperature of 60° C.
[0169] Due to low solubility of dNTP:Mg.sup.2+, it was not possible to perform the following experiments using magnesium complexed dNTPs alone.
[0170] DNA Amplification Reaction Setup
[0171] Reactions were set up at 500 μl scale as follows: A denaturation mix was prepared, and left at room temperature while the reaction mix was assembled. These were then mixed, and the DNA polymerase and pyrophosphatase added. DNA amplification experiments were then performed at a range of dNTP concentrations in a reaction buffer containing 60 mM Tris, pH 8.0. For sodium and ammonium complexed dNTPS (i.e. dNTP:4Na.sup.+ and dNTP:4NH.sub.4.sup.+) an equimolar amount of magnesium acetate was added to the reaction mix while for dNTPs complexed with sodium and magnesium or ammonium and magnesium, as described above, no additional magnesium was supplied to the reaction mix. Table 2 shows the experimental protocol for the DNA synthesis reactions.
[0172] The experiments were carried out to determine if reducing monovalent counterions on the dNTPS would result in higher dNTP usage. Reactions were grown for 168 hr (1 week) at a temperature of 30° C. followed by processing and quantification.
TABLE-US-00002 TABLE 2 DNA synthesis by Rolling Circle Amplification (RCA) Reaction components for examining the effects mono- and di-cation dNTP complexes. Stock Final reaction Reagent concentration Volume concentration Denaturation Plasmid Template 0.832 g/l 1.2 μl 2 ng/μl mix NaOH 1M 2.5 μl 5 mM DNA primer 5 mM 5 μl 50 μM H.sub.2O 18.8 μl to 25 μl Reaction Tris pH 8.0 1M 30 ul 60 mM mix dNTP salts 100 mM Variable Variable - as shown H.sub.2O Variable to 500 μl total Enzyme 1 Phi29 DNA 5.6 g/l 0.25 μl polymerase Enzyme 2 Pyrophosphatase 2000 U/ml 0.25 μl 0.4 U
[0173] Sample Processing Procedure
[0174] 222 mM EDTA was added after 168 hrs of RCA to a volume of 900 μl. 300 μl of water was added followed by mixing on “an end over end” rotary mixer over 4 hrs. 400 μl of 5M NaCl was added followed by 400 μl of 50% PEG 8000 (w/v). The reaction tubes were shaken vigorously for 15 minutes followed by further rotary mixing for 4 hrs. Precipitated DNA was recovered by centrifugation at 13,000 rpm in a bench-top centrifuge for 10 minutes. The supernatants were carefully decanted, and the pellets were resuspending in 9000 μl water overnight on an end over end mixer. Reaction DNA concentrations were quantified from UV absorption measurements on a nanodrop spectrophotometer. Data is corrected for the 18× fold increase in reaction volume and concentrations are expressed in g/l of original volume vs dNTP concentrations used.
[0175] Results
TABLE-US-00003 TABLE 3 DNA yields with different dNTP cation complexes at different concentrations. Peak yields are highlighted in bold Raw DNA yield (g/l) dNTPS: dNTP: dNTP: dNTP: 4Na.sup.+ 2Na•Mg.sup.2+ 4NH.sub.4.sup.+ 2NH.sub.4.sup.+•Mg.sup.2+ 10 mM 2.863 1.900 Not performed Not performed 20 mM 5.436 3.649 5.870 4.165 30 mM 7.611 4.938 9.57 8.402 40 mM 5.332 6.606 9.867 9.119 50 mM 1.214 7.817 3.997 9.569 60 mM 0.918 9.218 1.781 12.137 70 mM 0.926 10.164 1.597 12.042 80 mM 1.394 11.106 1.798 13.426
TABLE-US-00004 TABLE 4 DNA Efficiency with different dNTP cation complexes at different concentrations. Incorporation efficiency (%) dNTPS: dNTP: dNTP: dNTP: 4Na.sup.+ 2Na•Mg.sup.2+ 4NH.sub.4.sup.+ 2NH.sub.4.sup.+•Mg.sup.2+ 10 mM 88.1 58.5 Not performed Not performed 20 mM 83.6 56.1 90.3 64.1 30 mM 78.1 50.6 98.2 86.2 40 mM 41.0 50.8 75.9 70.1 50 mM 7.5 48.1 24.6 58.9 60 mM 4.7 47.3 9.1 62.2 70 mM 4.1 44.7 7.0 52.9 80 mM 5.4 42.7 6.9 51.6
[0176] The data in Tables 3 (represented graphically and also physically in
[0177] Compared to the corresponding sodium complexed dNTPs, the use of ammonium dNTP complexes (dNTP:4NH.sub.4.sup.+) results in peak production shifting higher to 40 mM dNTP concentrations producing raw yields of 9.867 g/l, almost two fold higher than the corresponding sodium counterbalanced dNTP (dNTP:4Na.sup.+).
[0178] However, the dNTP:2NH.sub.4.sup.+.Mg.sup.2+ complex has further increased usage to 80 mM and has resulted in a yield increase to at least 13.426 g/l. Also, as can be seem from
[0179] The figures demonstrating the viscosity of the reaction mixture once DNA has been synthesised are a very visual representation of the amount of DNA the process is able to synthesise. Once the DNA synthesis has taken place, the tubes have been inverted. Where no DNA or very little DNA is synthesised, the reaction mixture does not become viscous, and the reaction mixture collects at the cap of the tube. Where a sufficient amount of DNA is produced, the reaction mixture becomes very viscous, allowing the tube to be inverted and for the reaction mixture to remain in place in the tube. The more DNA, the more viscous the reaction mixture and the greater the retention in the tube. It can be seen that slightly less viscous products start to slip down the inside of the tubes once inverted.
Example 2
[0180] Effect of Different Monovalent Cations in Nucleotide Complexes with Magnesium on DNA Yield at a Range of Concentrations.
[0181] Materials and Methods
[0182] Reagents
[0183] The following reagents supplied were used in the presented examples: [0184] Solution 1-100 mM dATP: 4 K.sup.+ [0185] Solution 2-100 mM dCTP: 4 K.sup.+ [0186] Solution 3-100 mM dGTP: 4 K.sup.+ [0187] Solution 4-100 mM dTTP: 4 K.sup.+ [0188] Solution 5-100 mM dATP: 4 Cs.sup.+ [0189] Solution 6-100 mM dCTP: 4 Cs.sup.+ [0190] Solution 7-100 mM dGTP: 4 Cs.sup.+ [0191] Solution 8-100 mM dTTP: 4 Cs.sup.+ [0192] Solution 9-200 mM dATP: 4 NH.sub.4.sup.+ [0193] Solution 10-200 mM dCTP: 4 NH.sub.4.sup.+ [0194] Solution 11-200 mM dGTP: 4 NH.sub.4.sup.+ [0195] Solution 12-200 mM dTTP: 4 NH.sub.4.sup.+ [0196] Solution 13-66 mM dATP: 2 Mg.sup.2+ [0197] Solution 14-59 mM dCTP: 2 Mg.sup.2+ [0198] Solution 15-64 mM dGTP: 2 Mg.sup.2+ [0199] Solution 16-74 mM dTTP: 2 Mg.sup.2+ [0200] Phi29 DNA polymerase, stock concentration 5.6 g/L (produced in-house) [0201] Thermostable pyrophosphatase, stock concentration 2000 U/ml (Enzymatics) [0202] DNA primer, stock concentration 5 mM (Oligofactory) [0203] Plasmid template: ProTLx-K B5X4 LUX 15-0-15-10-15 AT-STEM, stock concentration 0.832 g/L (produced in house) [0204] Nuclease free water (Sigma Aldrich) [0205] 1M NaOH (Sigma Aldrich) [0206] PEG 8000 (Applichem) [0207] Tris-Base (Thermo Fisher Scientific) [0208] Tris-HCl (Sigma Aldrich) [0209] NaCl (Sigma Aldrich)
[0210] Preparation of dNTP Mixes
[0211] For the potassium (dATP: 4 K.sup.+), caesium (dATP: 4 Cs.sup.+) and ammonium (dATP: 4 NH.sub.4.sup.+) complexes the individual dNTPs (sol 1 to 12) were mixed 1:1:1:1 to produce a stock concentration of 100 mM dNTP mixture for potassium and caesium, while a 200 mM dNTP mixture for ammonium. The mixes were stored at −20° C.
[0212] For the magnesium mixed complexes (i.e. dNTPs: K.sup.+/Mg.sup.2+ or Cs.sup.+/Mg.sup.2+ or NH.sub.4.sup.+/Mg.sup.2+), dNTPs were mixed in such a way to provide an equimolar amount of each specific nucleotide (i.e. dATP, dCTP, dGTP & dTTP).
TABLE-US-00005 TABLE 5 Magnesium Potassium or Caesium dNTP Complex dNTP Complex [dXTP] Volume [dXTP] Volume dNTP mM (μl) dNTP mM (μl) dATP (sol 13) 66 1515 dATP (sol 1 or 5) 100 1000 dCTP (sol 14) 59 1695 dCTP (sol 2 or 6) 100 1000 dGTP (sol 15) 64 1563 dGTP (sol 3 or 7) 100 1000 dTTP (sol 16) 74 1351 dTTP (sol 4 or 8) 100 1000
TABLE-US-00006 TABLE 6 Magnesium dNTP Complex Ammonium dNTP Complex [dXTP] Volume [dXTP] Volume dNTP mM (μl) dNTP mM (μl) dATP (sol 13) 66 1515 dATP (sol 9) 200 500 dCTP (sol 14) 59 1695 dCTP (sol 10) 200 500 dGTP (sol 15) 64 1563 dGTP (sol 11) 200 500 dTTP (sol 16) 74 1351 dTTP (sol 12) 200 500
[0213] Magnesium nucleotides were mixed in such a way to provide equimolar amounts of each nucleotide. The volumes correspond to 100 mM of each nucleotide such that the final volume was 4000 μL.
[0214] Far caesium and potassium nucleotides, the final volume of 6124 μL was reduced to powder (i.e 0 μL) in a Speedvac at 60° C. The powder was resuspended by using the caesium or potassium pre-mixed nucleotides as detailed in Table 5, to a final volume of 4000 μL resulting in 200 mM K.sup.+/Mg.sup.2+ or Cs.sup.+/Mg.sup.2+ dNTPs.
[0215] For ammonium nucleotide, the final volume of 6124 μL was reduced to powder (i.e 0μL) in a Speedvac at 60° C. The powder was resuspended by using the ammonium pre-mixed nucleotides as detailed in Table 6 with an additional 2000 μl H.sub.2O to a final volume of 4000 μl resulting in 200 mM NH.sub.4.sup.+/Mg.sup.2+ dNTPs.
[0216] DNA Amplification Reaction Setup
[0217] Reactions were set up at 500 μl scale as follows: A denaturation mix was prepared and left at room temperature for 15 minutes while the reaction mix was assembled. These were then mixed, and the DNA polymerase and pyrophosphatase added. DNA amplification experiments were then performed at a range of dNTP concentrations with 30 mM pH 8.0 Tris buffer added. Reactions were split into 5 100 μl aliquots and stopped after 48, 72, 96, 120 and 144 hours. After stopping the samples were immediately processed as detailed below.
[0218] For mono-counterion complexed dNTPs (for example potassium dNTPs) an equimolar amount of magnesium chloride was added to the reaction mix. While for dNTPs complexed with both monovalent counterions (i.e. NH.sub.4.sup.+, K.sup.+, Cs.sup.+) and magnesium, as described above, no additional magnesium was supplied to the reaction mix. Table 5 shows the experimental protocol for the mono-complexed dNTPs (NH.sub.4.sup.+, K.sup.+& Cs.sup.+) reaction setup while Table 6 shows the reaction setup for the mixed dNTP complexes dNTPs (NH.sub.4.sup.+/Mg.sup.2+, K.sup.+/Mg.sup.2+& Cs.sup.+/Mg.sup.2+)
[0219] The experiments were carried out to determine if reducing monovalent counterions on the dNTPs would result in higher dNTP usage. Reactions were grown for the specified time period at a temperature of 30° C. followed by processing and quantification.
TABLE-US-00007 TABLE 7 DNA synthesis by Rolling Circle Amplification (RCA) Reaction components for examining the effects mono- and di-cation dNTP complexes. Stock Final reaction Reagent concentration Volume concentration Denaturation Plasmid Template 0.832 g/l 1.2 μL 2 ng/μL mix NaOH 1M 2.5 μL 5 mM DNA primer 5 mM 5 μL 50 μM H.sub.2O 18.8 μL to 25 μL Reaction mix Tris pH 8.0 1M 15 μL 30 mM Mono dNTP salts 100 mM Variable Variable - as shown MgCl.sub.2 2000 mM Variable Equimolar levels to dNTP molar concentrations H.sub.2O Variable to 500 μL total Enzyme 1 Phi29 5.6 g/L 0.25 μL DNA polymerase Enzyme 2 Pyrophosphatase 2000 U/mL 0.5 μL 1 U
TABLE-US-00008 TABLE 8 DNA synthesis by Rolling Circle Amplification (RCA. Reaction components for examining the effects mono- and di-cation dNTP complexes. Stock Final reaction Reagent concentration Volume concentration Denaturation Plasmid Template 0.832 g/l 1.2 μL 2 ng/μL mix NaOH 1M 2.5 μL 5 mM DNA primer 5 mM 5 μL 50 μM H.sub.2O 18.8 μL to 25 μL Reaction mix Tris pH 8.0 1M 15 μL 30 mM Mixed dNTP salts 200 mM Variable Variable - as shown H.sub.2O Variable to 500 μL total Enzyme 1 Phi29 5.6 g/L 0.25 μL DNA polymerase Enzyme 2 Pyrophosphatase 2000 U/mL 0.5 μL 1 U
[0220] Sample Processing Procedure
[0221] To each aliquot 900 μl of water was added for dilution and then 200 μl 5M NaCl and 500 μl of 25% PEG 8000 was added, solutions mixed by shaken vigorously for 15 minutes followed by further rotary mixing for 1 hour. The DNA was pelleted by centrifugation in a microcentrifuge (13,000 rpm, 30 minutes). The supernatants were carefully decanted, and the pellets were resuspended in 1000 μl water by vigorous shaking and air-displacement pipetting. Reaction DNA concentrations were quantified from UV absorption measurements on a nanodrop spectrophotometer at the end of the day and then left rotating overnight. Samples were re-checked in the morning and non reported any differences from previous day.
[0222] Data is corrected for the 10× fold increase in reaction volume and concentrations are expressed in g/l of original volume vs dNTP concentrations used. However, the high DNA yields may be underestimations due to the high difficulty in fully resuspending and solubilising very thick viscous DNA gels.
[0223] Results
TABLE-US-00009 TABLE 9 DNA yields with different dNTP cation complexes at different concentrations. Raw DNA Yield (g/l) dNTP: dNTP: dNTP: dNTP: dNTP: dNTP: 4K.sup.+ 2K.sup.+•Mg.sup.2+ 4NH.sub.4.sup.+ 2NH.sub.4.sup.+•Mg.sup.2+ 4Cs.sup.+ 2Cs.sup.+•Mg.sup.2+ 5 mM 1.65 1.11 1.24 1.10 1.18 1.12 10 mM 3.09 2.16 2.64 2.54 2.95 2.52 20 mM 5.52 4.65 5.18 3.98 5.69 3.88 30 mM 7.76 7.40 7.39 5.88 7.55 5.45 40 mM 1.41 7.60 9.78 9.26 7.65 7.68 80 mM 1.76 11.12 1.62 13.35 1.90 9.40 100 mM Not 2.31 1.99 15.89 Not 7.75 Performed Performed 120 mM Not 2.00 2.20 6.98 Not 2.15 Performed Performed
TABLE-US-00010 TABLE 10 DNA Efficiency with different dNTP cation complexes at different concentrations. Incorporation Efficiency (%) dNTP: dNTP: dNTP: dNTP: dNTP: dNTP: 4K.sup.+ 2K.sup.+•Mg.sup.2+ 4NH.sub.4.sup.+ 2NH.sub.4.sup.+•Mg.sup.2+ 4Cs.sup.+ 2Cs.sup.+•Mg.sup.2+ 5 mM 101.5% 68.3% 76.3% 67.7% 72.6% 68.9% 10 mM 95.1% 66.5% 81.2% 78.2% 90.8% 77.5% 20 mM 84.9% 71.5% 79.7% 61.2% 87.5% 59.7% 30 mM 79.6% 75.9% 75.8% 60.3% 77.4% 55.9% 40 mM 10.8% 58.5% 75.2% 71.2% 58.8% 59.1% 80 mM 6.8% 42.8% 6.2% 51.3% 7.3% 36.2% 100 mM Not 7.1% 6.1% 48.9% Not 23.8% Performed Performed 120 mM Not 5.1% 5.6% 17.9% Not 5.5% Performed Performed
[0224] The data in Table 9 demonstrates that by reducing the concentrations of monovalent counterions in dNTP complexes by the addition of magnesium cations, there is an increase in raw DNA produced. By using dNTPs with mixed counterions of a monovalent/divalent mixture i.e dNTP:2K.sup.+. Mg.sup.2+ the level of dNTP usage increases from 30 mM with standard dNTP:4K.sup.+ to at least 80 mM and the DNA yield from a peak of 7.76 g/l to 11.12 g/l respectively (Table 9).
[0225] Although the efficiency of conversion of dNTPs into DNA was lower for the dNTP:2K.sup.+.Mg.sup.2+ mix at lower concentrations compared to dNTP:4K.sup.+ decrease in overall efficiency was lower for dNTP:2K.sup.+.Mg.sup.2+ over the range of experimental conditions (Table 10).
[0226] By using dNTPs with mixed counterions of a monovalent/divalent mixture for ammonium dNTP:2NH.sub.4.sup.+.Mg.sup.2+ the level of dNTP usage increases from 40 mM with standard dNTP:4NH.sub.4.sup.+ to at least 100 mM and the DNA yield from a peak of 9.78 g/l to 15.89 g/l respectively (Table 9).
[0227] Although the efficiency of conversion of dNTPs into DNA was lower for the dNTP:2NH.sub.4.sup.+.Mg.sup.2+ mix at lower concentrations compared to dNTP:4NH.sub.4.sup.+ decrease in overall efficiency was lower for dNTP:2NH.sub.4.sup.+.Mg.sup.2+ over the range of experimental conditions (Table 10).
[0228] By using dNTPs with mixed counterions of a monovalent/divalent mixture for Caesium dNTP:2Cs.sup.+.Mg.sup.2+ the level of dNTP usage increases from 40 mM with standard dNTP:4Cs.sup.+ to at least 100 mM and the DNA yield from a peak of 7.65 g/l to 9.40 g/l respectively.
[0229] Although the efficiency of conversion of dNTPs into DNA was lower for the dNTP:2Cs.sup.+.Mg.sup.2+ mix at lower concentrations compared to dNTP:4Cs.sup.+ decrease in overall efficiency was lower for dNTP:2Cs.sup.+.Mg.sup.2+ over the range of experimental conditions (Table 10).
[0230] This experiment was essentially a repeat of Example 1 but using the monovalent cations potassium (K.sup.+) and caesium (Cs.sup.+) in place of sodium (Na.sup.+). Increased concentrations of nucleotides were tested up to 120 mM and reactions monitored over 2 to 6 days by measuring the total DNA produced. The experiment also included the use of ammonium cations (NH.sub.4.sup.+) repeating experiment 1 but increasing the concentrations of nucleotides to 120 mM.
[0231] DNA yields using nucleotide complexes dNTP:2K.sup.+.Mg.sup.2+, dNTP:2Cs.sup.+.Mg.sup.2+ and dNTP:2NH.sub.4.sup.+.Mg.sup.2+ were compared respectively with DNA yields obtained with the pure monovalent cation nucleotide complexes dNTP:4K.sup.+, dNTP:4Cs.sup.+ and dNTP:4NH.sub.4.sup.+ as controls. In the control experiments, an equimolar amount of magnesium (Mg.sup.2+) to nucleotide was provided in the form of the salt, magnesium chloride.
[0232]
[0233] Using these complexes compared to the control experiments not only reduces the concentration of monovalent cations in the reaction but also removes the anion on the magnesium salt. Thus, the ionic strength is significantly reduced.
Example 3
[0234] Effect of Different Salts of Magnesium on Magnesium Yields in the Control Experiments
[0235] This experiment was carried out to determine whether the nature of the magnesium salt could have a positive or detrimental effect on DNA yield in the control experiments. The magnesium salts compared were magnesium chloride and magnesium acetate since these are widely used in enzymatic DNA synthesis such as PCR.
[0236] Experiments were set up as in the previous examples with controls where magnesium was supplied in an equimolar concentration to the nucleotide using magnesium acetate and magnesium chloride. For reference, reactions using nucleotide complexes of sodium (dNTP:2Na.sup.+.Mg.sup.2+) and ammonium (dNTP:2NH.sub.4.sup.+.Mg.sup.2+) were also conducted.
[0237] The results in
Example 4
[0238] DNA Amplification Using a dNTP:2NH.sup.4+.Mg.sup.2+ Nucleotide Complex in the Absence of an External Buffering Agent
[0239] DNA Amplification Reaction Setup-Time Course
[0240] Reactions were set up at 1000 μl scale as follows: A denaturation mix was prepared, and left at room temperature while the reaction mix was assembled. These were then mixed, and the DNA polymerase and pyrophosphatase added. DNA amplification experiments were then performed at a range of dNTP concentrations with no additional buffering agents added. Reactions were split into 10×100 μl aliquots and incubated at 30° C. and stopped after 24, 48, 72, 96, 120, and 144 hrs by the addition of an Equimolar amount of EDTA to Mg.sup.2+. To each aliquot 25 μl of both 5M NaCl and 50% PEG 8000 was added, solutions mixed and DNA pelleted by centrifugation in a microcentrifuge (13,000 rpm, 15 minutes) The supernatants were carefully decanted, and the pellets were resuspending in 10000 μl water overnight on an end over end mixer. Reaction DNA concentrations were quantified from UV absorption measurements on a nanodrop spectrophotometer. Data is corrected for the 100× fold increase in reaction volume and concentrations are expressed in g/l of original volume vs dNTP concentrations used.
[0241] Reactions were set up as described previously to measure DNA amplification from a range of dNTP:2NH.sub.4.sup.+.Mg.sup.2+ nucleotide complex concentrations in the presence and absence of Tris HCl buffer. The control experiments were supplemented with Tris HCl buffer, pH 8.0 to give a final concentration of 30 mM while in the experimental group, Tris buffer was replaced by an equivalent volume of deionised water.
[0242] Individual reactions were set up at 100 μl scale and harvested for DNA measurement at daily intervals up to 6 days. Starting concentrations of the dNTP:2NH.sub.4.sup.+.Mg.sup.2+ nucleotide complex ranged from 25 mM to 125 mM.
[0243] The results in