Nucleic acid amplifications
A method includes combining a polynucleotide and an amplification reagent mixture to form a reaction mixture, wherein the reaction mixture comprises reversibly bound divalent ions in solution, and adjusting the pH of the reaction mixture to release the reversibly bound divalent ions, thereby initiating amplification of the polynucleotide.
1. A method comprising:
(a) combining a polynucleotide and an amplification reagent mixture at a first temperature between 10° C. and 30° C. to form a reaction mixture, wherein the amplification reagent mixture comprises a polymerase, magnesium ions, a temperature-sensitive buffer, and a multidentate pH-sensitive chelating agent, wherein the ratio of the multidentate pH-sensitive chelating agent concentration to the magnesium ion concentration is from about 0.5 to about 2;
(b) adjusting the temperature of the reaction mixture from
(i) said first temperature at which the pH of the temperature-sensitive buffer is operable for the pH-sensitive chelating agent to reversibly bind free magnesium ions in solution, such that amplification of the polynucleotide is inhibited, to
(ii) a second temperature between 40° C. and 70° C. at which the pH of the temperature-sensitive buffer is operable to release bound magnesium ions from the pH-sensitive chelating agent, such that amplification of the polynucleotide can proceed; and
(c) amplifying the polynucleotide at said second temperature without repeated cycling of the temperature of the reaction mixture between the first temperature and the second temperature.
2. The method of claim 1 , wherein the reaction mixture comprises a free magnesium ion concentration at the first temperature that is between about 0 and about 10 mM.
3. The method of claim 1 , wherein the reaction mixture comprises a free magnesium ion concentration at the first temperature that is between about 0 and about 5 mM.
4. The method of claim 1 , wherein the reaction mixture comprises a free magnesium ion concentration at the first temperature that is between about 0 and about 2 mM.
5. The method of claim 1 , wherein the reaction mixture comprises a free magnesium ion concentration at the first temperature that is between about 0 and about 1 mM.
6. The method of claim 1 , wherein the reaction mixture comprises a free magnesium ion concentration at the first temperature that is between about 0 and about 0.2 mM.
7. The method of claim 1 , wherein the reaction mixture comprises a free magnesium ion concentration at the first temperature that is between about 0 and about 0.1 mM.
8. The method of claim 1 , wherein the reaction mixture comprises a free magnesium ion concentration at the second temperature that is between about 5 mM and about 50 mM.
9. The method of claim 1 , wherein the reaction mixture comprises a free magnesium ion concentration at the second temperature that is between about 10 mM and about 50 mM.
10. The method of claim 1 , wherein the reaction mixture comprises a free magnesium ion concentration at the second temperature that is between about 10 mM and about 20 mM.
11. The method of claim 1 , wherein the reaction mixture comprises a free magnesium ion concentration at the second temperature that is between about 5 mM and about 20 mM.
12. The method of claim 1 , wherein the reaction mixture comprises a free magnesium ion concentration at the second temperature that is between about 5 mM and about 10 mM.
13. The method of claim 1 , wherein the multidentate pH-sensitive chelating agent is selected from the group consisting of ethyleneglycol-bis(2-aminoethylether) tetraacetic acid, EGTA derivatives, and EDTA derivatives.
14. The method of claim 1 , wherein the pKa of the temperature-sensitive buffer at the second temperature is at least 0.4 less than the pKa of the temperature-sensitive buffer at the first temperature.
15. The method of claim 1 , wherein the temperature-sensitive buffer comprises tris(hydroxymethyl)aminomethane.
16. The method of claim 1 , wherein the first temperature is between 10° C. and about 20° C. or between about 20° C. and 30° C.
17. The method of claim 1 , wherein the first temperature is between about 20° C. and 30° C.
18. The method of claim 1 , wherein the second temperature is between 40° C. and about 50° C., between about 50° C. and about 60° C., or between about 60° C. and 70° C.
19. The method of claim 1 , wherein the second temperature is between about 60° C. and 70° C.
20. The method of claim 1 , wherein the amplification reagent mixture comprises a nicking endonuclease.
21. The method of claim 1 , wherein the amplification reagent mixture comprises a DNA polymerase or an RNA polymerase.
22. The method of claim 1 , wherein the amplification reagent mixture comprises a reverse transcriptase.
23. The method of claim 1 , wherein the amplification reagent mixture comprises:
(1) a first oligonucleotide comprising a 5′ portion that comprises a nicking site and that is non-complementary to a target sequence of the polynucleotide and a 3′ portion that hybridizes to the target sequence of the polynucleotide; and
(2) a second oligonucleotide comprising a 5′ portion that comprises a nicking site and that is non-complementary to the target sequence of the polynucleotide and a 3′ portion that hybridizes to the target sequence of the polynucleotide.
24. The method of claim 1 , wherein amplification of the polynucleotide occurs under substantially isothermal conditions.
25. The method of claim 1 , wherein the polynucleotide is not denatured prior to combining with the amplification reagent mixture.
26. The method of claim 1 , further comprising:
(d) detecting amplified polynucleotides.
27. The method of claim 1 , wherein one or more components of the amplification reagent mixture is provided in a container suitable for use in a fluidic device, cartridge, or lateral flow device.
28. A method comprising:
(a) combining a polynucleotide and an amplification reagent mixture at a first temperature between 10° C. and 30° C. to form a reaction mixture, wherein the amplification reagent mixture comprises a polymerase, magnesium ions, a temperature sensitive buffer, and a multidentate pH-sensitive chelating agent selected from the group consisting of EDTA, EDTA derivatives, EGTA, and EGTA derivatives, wherein the ratio of the multidentate pH-sensitive chelating agent concentration to the magnesium ion concentration is from about 0.5 to about 2;
(b) adjusting the temperature of the reaction mixture from
(i) said first temperature at which the pH of the temperature-sensitive buffer is operable for the multidentate pH-sensitive chelating agent to reversibly bind free magnesium ions from solution, such that amplification of the polynucleotide is inhibited, to
(ii) a second temperature between 40° C. and 70° C. at which the pH of the temperature-sensitive buffer is operable to release bound magnesium ions from the multidentate pH-sensitive chelating agent to solution, such that amplification of the polynucleotide can proceed; and
(c) amplifying the polynucleotide at said second temperature.