nucleic acid amplification
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- IONIAN TECHNOLOGIES LLC
- Filing Date
- 2026-03-06
- Publication Date
- 2026-06-16
Smart Images
Figure 2026097977000001_ABST
Abstract
Claims
1. (a) A step of combining a polynucleotide and an amplification reagent mixture to form a reaction mixture, wherein the reaction mixture contains divalent ions that are reversibly bound in solution, (b) Adjust the pH of the reaction mixture to release the reversibly bound divalent ions, This initiates the step of amplifying polynucleotides. A method that includes this.
2. The method according to claim 1, wherein the divalent ion is selected from the group consisting of magnesium, calcium, copper, zinc, manganese, iron, cadmium, and lead.
3. The method according to claim 1 or claim 2, wherein the divalent ion is magnesium.
4. The method according to any one of claims 1 to 3, wherein the amplification reagent mixture comprises a pH-sensitive chelating agent.
5. The method according to claim 4, wherein the pH-sensitive chelating agent is selected from the group consisting of ethylene glycol-bis(2-aminoethyl ether)tetraacetic acid, EGTA derivatives, and EDTA derivatives.
6. The method according to any one of claims 1 to 5, wherein the amplification reagent mixture comprises a temperature-sensitive buffer.
7. The method according to claim 6, wherein the temperature-sensitive buffer comprises tris(hydroxymethyl)aminomethane.
8. The method according to claim 6 or 7, wherein the pH of the reaction mixture is adjusted according to the pH of the temperature-sensitive buffer.
9. The method according to any one of claims 1 to 8, wherein the pH of the reaction mixture is adjusted by changing the temperature of the reaction mixture from a first temperature to a second temperature.
10. The method according to claim 9, wherein the pKa of the temperature-sensitive buffer at the second temperature is at least 0.4 lower than the pKa of the temperature-sensitive buffer at the first temperature.
11. Temperature-sensitive buffer at -0.04°C －１ ~-0.015℃ －１ The method according to any one of claims 6 to 10, having ΔpKa.
12. The method according to any one of claims 9 to 11, wherein the first temperature is about 0°C to about 10°C, about 10°C to about 20°C, or about 20°C to about 30°C.
13. The method according to any one of claims 9 to 12, wherein the second temperature is approximately 30°C to approximately 40°C, approximately 40°C to approximately 50°C, approximately 50°C to approximately 60°C, approximately 60°C to approximately 70°C, approximately 70°C to approximately 80°C, approximately 80°C to approximately 90°C, or approximately 90°C to approximately 100°C.
14. The method according to any one of claims 1 to 13, wherein the amplification reagent mixture comprises a nickel endonuclease.
15. The method according to any one of claims 1 to 14, wherein the amplification reagent mixture comprises DNA or RNA polymerase.
16. The method according to any one of claims 1 to 15, wherein the amplification reagent mixture comprises reverse transcriptase.
17. The method according to any one of claims 1 to 16, wherein the ratio of the chelating agent concentration to the divalent ion concentration is about 0.5 to about 2.
18. The method according to any one of claims 9 to 17, wherein the concentration of free divalent ions at a first temperature is about 0 to about 10 mM.
19. The method according to any one of claims 9 to 18, wherein the concentration of free divalent ions at the second temperature is about 5 mM to about 50 mM.
20. The method according to any one of claims 1 to 19, wherein the amplification reagent mixture comprises one or more components in a lyophilized form.
21. The method according to claim 20, wherein the amplification reagent mixture comprises a magnesium salt in a lyophilized form.
22. The method according to claim 20, wherein the amplification reagent mixture comprises a pH-sensitive chelating agent in lyophilized form.
23. The method according to claim 21, wherein a freeze-dried magnesium salt is reconstituted in a buffer to form magnesium ions in solution.
24. The method according to claim 22, wherein a lyophilized pH-sensitive chelating agent is reconstituted in a buffer.
25. The method according to claim 23 or 24, wherein magnesium ions in the solution reversibly bind to a pH-sensitive chelating agent.
26. The method according to claim 23 or 24, wherein the buffer is a temperature-sensitive buffer.
27. The method according to any one of claims 23 to 26, wherein the pH of the buffer is operable so that a pH-sensitive chelating agent reversibly binds to free magnesium ions in the solution.
28. The method according to any one of claims 1 to 27, wherein the amplification of polynucleotides occurs under substantially isothermal conditions.
29. The method according to any one of claims 1 to 28, wherein the polynucleotide is not denatured before being combined with the amplification reagent mixture.
30. The method according to any one of claims 1 to 29, wherein the combining step is carried out at a temperature of approximately 0°C to approximately 10°C, approximately 10°C to approximately 20°C, or approximately 20°C to approximately 30°C.
31. The method according to any one of claims 1 to 30, wherein the amplification of the polynucleotide does not occur until the temperature of the reaction mixture reaches about 30°C to about 40°C, about 40°C to about 50°C, about 50°C to about 60°C, about 60°C to about 70°C, about 70°C to about 80°C, about 80°C to about 90°C, or about 90°C to about 100°C.
32. The method according to any one of claims 1 to 31, wherein the amplification of a polynucleotide occurs without repeated temperature cycles of the reaction mixture between a first temperature and a second temperature.
33. The method according to any one of claims 1 to 32, wherein one or more components of the amplification reagent mixture are provided in a container suitable for use in a fluid device, cartridge, or lateral flow device.
34. The method according to any one of claims 1 to 33, wherein the amplification of the polynucleotide occurs without adding any further reagents to the reaction mixture formed in step (a) of combining.
35. The method according to any one of claims 1 to 34, further comprising the step (c) of detecting amplified polynucleotides.
36. The method according to claim 35, wherein the detection of the amplified polynucleotide occurs without adding any further reagents to the reaction mixture formed in step (a) of combining.
37. The method according to any one of claims 1 to 36, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially all of the divalent ions in the reaction mixture are in a soluble form.
38. The method according to claim 37, wherein a divalent ion reversibly binds to a pH-sensitive chelating agent.
39. The method according to claim 37, wherein the divalent ion includes a free divalent ion.
40. The method according to claim 37, comprising both a divalent ion bound to a divalent ion and a free divalent ion.
41. The method according to any one of claims 1 to 40, wherein divalent ions in the solution are not formed from the dissolution of the precipitate.
42. The method according to any one of claims 1 to 41, wherein, prior to the amplification of the polynucleotide, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the divalent ions form a precipitate, or substantially none form a precipitate.
43. The method according to claim 35, wherein, prior to the detection of the amplified polynucleotide, less than 20%, less than 15%, less than 10%, less than 5%, and less than 1% of the divalent ions form a precipitate or substantially none form a precipitate.
44. The method according to any one of claims 1 to 43, wherein the reaction mixture does not contain divalent ions bound in a precipitated form.
45. (a) A step of forming a reaction mixture by combining a polynucleotide and an amplification reagent mixture, wherein the amplification reagent mixture comprises magnesium ions reversibly bound in solution, a temperature-sensitive buffer and a pH-sensitive chelating agent, (b) The temperature of the reaction mixture (i) From a first temperature at which the pH of the temperature-sensitive buffer is operable so that the pH-sensitive chelating agent reversibly binds to free magnesium ions in the solution so that the amplification of polynucleotides is inhibited, (ii) Adjust the pH of the temperature-sensitive buffer to a second temperature at which it is operable so that bound magnesium ions are released from the pH-sensitive chelating agent, so that the amplification of polynucleotides can proceed. This initiates the step of amplifying polynucleotides. A method that includes this.
46. The method according to claim 45, wherein the pH-sensitive chelating agent is selected from the group consisting of ethylene glycol-bis(2-aminoethyl ether)tetraacetic acid, EGTA derivatives, and EDTA derivatives.
47. The method according to claim 45 or 46, wherein the temperature-sensitive buffer comprises tris(hydroxymethyl)aminomethane.
48. The method according to any one of claims 45 to 47, wherein the pKa of the temperature-sensitive buffer at the second temperature is at least 0.4 lower than the pKa of the temperature-sensitive buffer at the first temperature.
49. Temperature-sensitive buffer at -0.04°C －１ ~-0.015℃ －１ The method according to any one of claims 45 to 48, having ΔpKa.
50. The method according to any one of claims 45 to 49, wherein the first temperature is about 0°C to about 10°C, about 10°C to about 20°C, or about 20°C to about 30°C.
51. The method according to any one of claims 45 to 50, wherein the second temperature is approximately 30°C to approximately 40°C, approximately 40°C to approximately 50°C, approximately 50°C to approximately 60°C, approximately 60°C to approximately 70°C, approximately 70°C to approximately 80°C, approximately 80°C to approximately 90°C, or approximately 90°C to approximately 100°C.
52. The method according to any one of claims 45 to 51, wherein the amplification reagent mixture comprises a nickel endonuclease.
53. The method according to any one of claims 45 to 52, wherein the amplification reagent mixture comprises DNA or RNA polymerase.
54. The method according to any one of claims 45 to 53, wherein the amplification reagent mixture comprises a reverse transcriptase.
55. The method according to any one of claims 45 to 54, wherein the ratio of the chelating agent concentration to the magnesium ion concentration is about 0.5 to about 2.
56. The method according to any one of claims 45 to 55, wherein the concentration of free magnesium ions at a first temperature is about 0 to about 10 mM.
57. The method according to any one of claims 45 to 56, wherein the concentration of free magnesium ions at the second temperature is about 5 mM to about 50 mM.
58. The method according to any one of claims 45 to 57, wherein the amplification reagent mixture comprises one or more components in a lyophilized form.
59. The method according to any one of claims 45 to 58, wherein the amplification reagent mixture comprises a magnesium salt in a lyophilized form.
60. The method according to any one of claims 45 to 59, wherein the amplification reagent mixture comprises a pH-sensitive chelating agent in lyophilized form.
61. The method according to claim 59, wherein a freeze-dried magnesium salt is reconstituted in a buffer to form magnesium ions in solution.
62. The method according to claim 60, wherein a lyophilized pH-sensitive chelating agent is reconstituted in a buffer.
63. The method according to claim 61, wherein magnesium ions in the solution reversibly bind to a pH-sensitive chelating agent.
64. The method according to claim 61 or 62, wherein the buffer is a temperature-sensitive buffer.
65. The method according to any one of claims 61 to 64, wherein the pH of the buffer is operable so that a pH-sensitive chelating agent reversibly binds to free magnesium ions in the solution.
66. The method according to any one of claims 45 to 65, wherein the amplification of polynucleotides occurs under substantially isothermal conditions.
67. The method according to any one of claims 45 to 66, wherein the polynucleotide is not denatured before being combined with the amplification reagent mixture.
68. The method according to any one of claims 45 to 67, wherein the combining step is carried out at a temperature of approximately 0°C to approximately 10°C, approximately 10°C to approximately 20°C, or approximately 20°C to approximately 30°C.
69. The method according to any one of claims 45 to 68, wherein the amplification of the polynucleotide does not occur until the temperature of the reaction mixture reaches about 30°C to about 40°C, about 40°C to about 50°C, about 50°C to about 60°C, about 60°C to about 70°C, about 70°C to about 80°C, about 80°C to about 90°C, or about 90°C to about 100°C.
70. The method according to any one of claims 45 to 69, wherein the amplification of the polynucleotide occurs without repeated temperature cycles of the reaction mixture between a first temperature and a second temperature.
71. The method according to any one of claims 45 to 70, wherein one or more components of the amplification reagent mixture are provided in a container suitable for use in a fluid device, cartridge, or lateral flow device.
72. The method according to any one of claims 45 to 71, wherein the amplification of the polynucleotide occurs without adding any further reagents to the reaction mixture formed in step (a) of combining.
73. The method according to any one of claims 45 to 72, further comprising the step (c) of detecting amplified polynucleotides.
74. The method according to claim 73, wherein the detection of the amplified polynucleotide occurs without adding any further reagents to the reaction mixture formed in step (a) of combining.
75. The method according to any one of claims 45 to 74, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially all magnesium ions in the reaction mixture are in a soluble form.
76. The method according to claim 75, wherein magnesium ions are reversibly bound to a pH-sensitive chelating agent.
77. The method according to claim 75, wherein the magnesium ions include free magnesium ions.
78. The method according to claim 75, wherein the magnesium ions include both bound magnesium ions and free magnesium ions.
79. The method according to any one of claims 45 to 78, wherein magnesium ions in the solution are not formed from the dissolution of the precipitate.
80. The method according to any one of claims 45 to 79, wherein, prior to the amplification of the polynucleotide, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1% of the magnesium ions form a precipitate or substantially none form a precipitate.
81. The method according to claim 73, wherein, prior to the detection of the amplified polynucleotide, less than 20%, less than 15%, less than 10%, less than 5%, and less than 1% of the magnesium ions form a precipitate or substantially none form a precipitate.
82. The method according to any one of claims 45 to 81, wherein the reaction mixture does not contain magnesium ions bound in a precipitated form.
83. A composition comprising one or more reagents for nucleic acid amplification and pH-dependent, reversibly bound magnesium ions in solution.
84. The composition according to claim 83, further comprising a temperature-sensitive buffer and a pH-sensitive chelating agent.
85. The composition according to claim 84, wherein the temperature-sensitive buffer comprises tris(hydroxymethyl)aminomethane.
86. Temperature-sensitive buffer at -0.04°C －１ ~-0.015℃ －１ The composition according to claim 84 or 85, having a ΔpKa of .
87. The composition according to any one of claims 84 to 86, wherein the pH-sensitive chelating agent is ethylene glycol-bis(2-aminoethyl ether)tetraacetic acid.
88. The composition according to any one of claims 83 to 87, wherein one or more reagents for nucleic acid amplification comprises a nickel endonuclease.
89. The composition according to any one of claims 83 to 88, wherein one or more reagents for nucleic acid amplification comprises a DNA or RNA polymerase.
90. The composition according to any one of claims 83 to 89, wherein one or more reagents for nucleic acid amplification include a reverse transcriptase.
91. The composition according to any one of claims 83 to 90, wherein one or more reagents for nucleic acid amplification include a recombinase.
92. The composition according to any one of claims 83 to 91, wherein one or more reagents for nucleic acid amplification comprises one or more components in a lyophilized form.
93. The composition according to any one of claims 83 to 92, wherein one or more reagents for nucleic acid amplification comprises a magnesium salt in lyophilized form.
94. The composition according to any one of claims 83 to 93, wherein one or more reagents for nucleic acid amplification comprises a pH-sensitive chelating agent in lyophilized form.
95. The composition according to claim 93, wherein a freeze-dried magnesium salt is reconstituted in a buffer to form magnesium ions in solution.
96. The composition according to claim 94, wherein a lyophilized pH-sensitive chelating agent is reconstituted in a buffer.
97. The composition according to claim 95 or 96, wherein the buffer is a temperature-sensitive buffer.
98. The composition according to any one of claims 83 to 97, wherein magnesium ions in the solution are not formed from the dissolution of precipitates.
99. The composition according to any one of claims 83 to 98, which does not contain magnesium ions bound in a precipitated form.
100. The composition according to any one of claims 83 to 99, wherein one or more components are provided in a container suitable for use in a fluid device, cartridge, or lateral flow device.
101. A composition comprising one or more reagents for nucleic acid amplification, a temperature-sensitive buffer, a pH-sensitive chelating agent, and a magnesium salt.
102. The composition according to claim 101, wherein the temperature-sensitive buffer comprises tris(hydroxymethyl)aminomethane.
103. Temperature-sensitive buffer at -0.04°C －１ ~-0.015℃ －１ The composition according to claim 101 or 102, having a ΔpKa of .
104. The composition according to any one of claims 101 to 103, wherein the pH-sensitive chelating agent is ethylene glycol-bis(2-aminoethyl ether)tetraacetic acid.
105. The composition according to any one of claims 101 to 104, wherein one or more reagents for nucleic acid amplification include a nickel endonuclease.
106. The composition according to any one of claims 101 to 105, wherein one or more reagents for nucleic acid amplification include a DNA or RNA polymerase.
107. The composition according to any one of claims 101 to 106, wherein one or more reagents for nucleic acid amplification include a reverse transcriptase.
108. The composition according to any one of claims 101 to 107, wherein one or more reagents for nucleic acid amplification include a recombinase.
109. The composition according to any one of claims 101 to 108, wherein one or more reagents for nucleic acid amplification comprises one or more components in a lyophilized form.
110. The composition according to any one of claims 101 to 109, wherein the magnesium salt is in a freeze-dried form.
111. The composition according to any one of claims 101 to 110, wherein the pH-sensitive chelating agent is in a freeze-dried form.
112. The composition according to claim 110, wherein a freeze-dried magnesium salt is reconstituted in a buffer to form magnesium ions in a solution.
113. The composition according to claim 111, wherein a lyophilized pH-sensitive chelating agent is reconstituted in a buffer.
114. The composition according to claim 112 or 113, wherein the buffer is a temperature-sensitive buffer.
115. The composition according to claim 112, wherein magnesium ions in the solution are not formed from the dissolution of precipitates.
116. The composition according to any one of claims 101 to 115, which does not contain magnesium ions bound in a precipitated form.
117. The composition according to any one of claims 101 to 116, wherein one or more components are provided in a container suitable for use in a fluid device, cartridge, or lateral flow device.
118. (a) A step of combining an enzyme and a reagent mixture to form a reaction mixture, wherein the reaction mixture contains divalent ions that are reversibly bound in solution, (b) Adjust the pH of the reaction mixture to release the reversibly bound divalent ions, This involves the step of activating the enzyme and A method that includes this.
119. The method according to claim 118, wherein the divalent ion is selected from the group consisting of magnesium, calcium, copper, zinc, manganese, iron, cadmium, and lead.
120. The method according to claim 118 or 119, wherein the reagent mixture comprises a pH-sensitive chelating agent.
121. The method according to any one of claims 118 to 120, wherein the reagent mixture comprises a temperature-sensitive buffer.
122. The method according to claim 121, wherein the pH of the reaction mixture is adjusted according to the pH of the temperature-sensitive buffer.
123. The method according to any one of claims 118 to 122, wherein the pH of the reaction mixture is adjusted by changing the temperature of the reaction mixture from a first temperature to a second temperature.
124. The method according to any one of claims 118 to 123, wherein the enzyme is a DNA or RNA polymerase.
125. The method according to any one of claims 118 to 123, wherein the enzyme is a nickel end nuclease.