CN-121992052-A - Synthesis of DNA with increased yield

Abstract

The present invention relates to an improved method for synthesizing deoxyribonucleic acid (DNA), in particular cell-free enzymatic synthesis of DNA, preferably on a large scale, with improved yield and/or improved efficiency. The cationic species present as counter-ion in the nucleotide salts is critical to the yield, efficiency and accuracy of high yield enzymatic DNA synthesis reactions. The methods herein use alternative cations as counter ions for ionic nucleotides, allowing for the use of higher concentrations of nucleotides in DNA synthesis and further allowing for the use of more favorable reaction conditions.

Inventors

• NEIL PORTER
• Paul James Roswell
• S. Grick janini
• A. Babula
• T. A.J. Eddie

Assignees

• 塔驰莱特IP有限公司

Dates

Publication Date

20260508

Application Date

20190816

Priority Date

20180817

Claims (20)

1. 1. A cell-free process for enzymatically synthesizing DNA comprising the use of a nucleotide salt, wherein the salt comprises a monovalent cation having an ionic radius greater than the ionic radius of sodium ions.
2. 2. The cell-free process of claim 1, wherein the nucleotide salt is present at a concentration of greater than 10 mM.
3. 3. A cell-free process for enzymatically synthesizing DNA, comprising using a nucleotide salt, wherein the nucleotide salt is present at a concentration of at least 10mM, and is: (a) A nucleotide salt comprising a monovalent cation having an ionic radius greater than that of sodium ion, or (B) Two or more nucleotide salts, each salt comprising a different monovalent cation, wherein at least one cation has an ionic radius greater than the ionic radius of the sodium ion.
4. 4. The cell-free process of any preceding claim, wherein the nucleotide salt is present at a concentration of at least 15mM, at least 20mM, at least 25mM, at least 30mM, at least 35mM, or at least 40 mM.
5. 5. The cell-free process of any preceding claim, wherein the one or more monovalent cations are independently selected from alkaline earth metals, transition metals, or polyatomic ions.
6. 6. The cell-free process of claim 5, wherein the one or more monovalent cations are independently selected from the list comprising potassium, ammonium, derivatives of ammonium, rubidium, cesium, or francium.
7. 7. The cell-free process of any preceding claim, wherein the cell-free process further comprises the use of one or more primers or primer enzymes.
8. 8. The cell-free process of any preceding claim, wherein the cell-free process further comprises using a template.
9. 9. The cell-free process for synthesizing DNA according to any preceding claim, wherein the cell-free process further comprises the use of one or more divalent metal cations, preferably selected from the list comprising magnesium, manganese, calcium, beryllium, zinc and strontium.
10. 10. The cell-free process of claim 9, wherein the ratio of divalent metal cations to nucleotides in the reaction mixture is equal to or less than 1:1, preferably less than 1:1.
11. 11. The cell-free process of any preceding claim, wherein the process uses sodium and/or lithium salts of nucleotides at a maximum concentration of 10 mM.
12. 12. The cell-free process of any preceding claim, wherein the process further comprises the use of a chemical denaturant, preferably sodium hydroxide, potassium hydroxide or ammonium hydroxide, and pyrophosphatase.
13. 13. A 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. 14. The cell-free process of claim 13, wherein the nucleotide salt comprises cesium ions.
15. 15. The cell-free process of claim 12, wherein a pH buffer is added, but no additional salt or detergent is added.
16. 16. The cell-free process of claim 15, wherein the nucleotide salt comprises an ammonium ion.
17. 17. The cell-free process according to any preceding claim, wherein the process is used for large scale synthesis of DNA, preferably at least 3g/l.
18. 18. Use of a nucleotide comprising cesium cations in enzymatic cell-free synthesis of DNA.
19. 19. Use according to claim 18, wherein enzymatic cell-free synthesis of DNA is performed in the presence of low levels of divalent cations, optionally in a ratio of divalent cations to nucleotides of 0.2:1 to 0.8:1, preferably 0.2:1 to 0.5:1.
20. 20. The use of claim 18, wherein cell-free synthesis of DNA is performed in minimal buffer, optionally comprising only pH buffer, no detergent or additional salts.

Description

Synthesis of DNA with increased yield The present application is a divisional application of the title of "synthesizing DNA with improved yield" with application number 2019800678114, 16 of 8 th 2019. Technical Field The present invention relates to an improved method for synthesizing deoxyribonucleic acid (DNA), in particular cell-free enzymatic synthesis of DNA, preferably on a large scale, with improved yield and/or improved efficiency. Background Amplification of deoxyribonucleic acid (DNA) may be performed by using cell-based methods, such as by culturing bacteria that proliferate the DNA to be amplified in a fermenter. Cell-free enzymatic methods for amplifying DNA from a starting template have also been described, including polymerase chain reaction and strand displacement reaction. In the past, test-scale DNA amplification has been performed using devices based on microtiter plates and automatically controlled pipettes to add the required reaction components. Such devices and methods are suitable for producing small amounts of DNA molecules for testing purposes, but do not provide sufficient amounts for other purposes. Large scale amplification and production of specific nucleic acids and proteins is largely carried out by cell-based methods. Such a method is generally effective for the production of very large quantities of products, but is costly to manufacture. In addition, for clinical and therapeutic purposes, it is preferred to synthesize DNA in a cell-free environment. Large-scale DNA synthesis using chemical synthesis, such as the phosphoramidite method, is known, but is not without drawbacks. The reaction must generally be carried out in organic solvents, many of which are toxic or otherwise detrimental. Another disadvantage of chemical synthesis is that it is not entirely efficient, since after each addition of a nucleotide a percentage of the growing oligonucleotide chains are capped, resulting in yield loss. Thus, the total yield loss of the synthesized nucleotide chain increases with each nucleotide added to the sequence. This inherent inefficiency of chemically synthesizing oligonucleotides ultimately limits the length of oligonucleotides that can be efficiently produced to oligonucleotides having 50 or fewer nucleic acid residues, and further affects the accuracy of the synthesis. Heretofore, biocatalysts such as polymerases have not been routinely developed for the production of DNA products on an in vitro industrial scale, and the reaction is largely limited to microliter scale volumes. Amplification methods using enzymatically synthesized DNA have proven problematic, especially in terms of disappointing yields of DNA products. The inventors have previously addressed the ability to amplify using commercially available nucleotides. As described in WO2016/034849, a new method was developed which involved adding fresh nucleotides to the reaction mixture when the fresh nucleotides were depleted or the product concentration reached a threshold value, which is incorporated herein by reference. It has been determined that even higher yields can be achieved and the inventors have developed the novel methods described herein to further increase the yield of enzymatic DNA synthesis. Enzymatic DNA synthesis typically requires the use of a polymerase or polymerase-like enzyme to catalyze the addition of nucleotides to nascent nucleic acid strands. In general, template DNA amplified in the reaction is required. However, template-free DNA synthesis may also be performed, wherein integration is performed de novo. It is important to note that due to the highly charged nature of nucleic acids, they are often surrounded by counter ions to neutralize most of their charge to reduce electrostatic repulsion between parts of the sequence, thus enabling them to be condensed into a neat, compact structure in the cell. Nucleic acid building blocks (nucleotides) are also ionic substances that require the presence of positive counterions to maintain electroneutrality. Thus, most, if not all, of the nucleotides are provided as salts with positive counterions. Since nucleotides have four negative charges, salts are typically prepared with 2 divalent cations or 4 monovalent cations. It will be apparent to those skilled in the art that once the nucleotide salt is dispersed in water or other solvent, the salt can dissociate into anionic and cationic components in solution. Typically, the nucleotides are provided in the form of lithium or sodium salts for DNA synthesis, amplification or sequencing. Lithium is generally preferred because of its greater solubility and stability to repeated freeze-thaw cycles than sodium salts, and because of its bacteriostatic activity against various microorganisms, it remains sterile, providing greater reliability and longer shelf life. The use of these salts is so conventional that the person skilled in the art seems to have no question about the counter-ion present with the nucleo