Nucleic acid amplifications

Abstract

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 - 28 . (canceled) 
     
     
         29 . An amplification reagent mixture comprising a nucleic acid amplification reagent and pH-dependent reversibly bound magnesium ions. 
     
     
         30 . The amplification reagent mixture of  claim 29 , wherein the nucleic acid amplification reagent comprises a nicking endonuclease, a DNA polymerase, an RNA polymerase, a recombinase, and/or a reverse transcriptase. 
     
     
         31 . The amplification reagent mixture of  claim 29 , further comprising a temperature-sensitive buffer. 
     
     
         32 . The amplification reagent mixture of  claim 31  wherein the temperature-sensitive buffer has a ΔpK a  of between −0.040° C. −1  and −0.015° C. −1 . 
     
     
         33 . The amplification reagent mixture of  claim 29 , further comprising a multidentate pH-sensitive chelating agent. 
     
     
         34 . The amplification reagent mixture of  claim 33 , wherein said multidentate pH-sensitive chelating agent comprises ethyleneglycol-bis(2-aminoethylether) tetraacetic acid (EGTA), an EGTA derivative, or an ethylenediaminetetraacetic acid (EDTA) derivative. 
     
     
         35 . The amplification reagent mixture of  claim 33 , wherein the ratio of the multidentate pH-sensitive chelating agent concentration to the magnesium ion concentration is from about 0.5 to about 2.0. 
     
     
         36 . The amplification reagent mixture of  claim 29 , further comprising a polynucleotide. 
     
     
         37 . The amplification reagent mixture of  claim 29 , wherein said amplification reagent mixture has a temperature between 10° C. and 30° C. 
     
     
         38 . The amplification reagent mixture of  claim 29 , further comprising free magnesium ions. 
     
     
         39 . The amplification reagent mixture of  claim 29 , further comprising between 0 mM and 10 mM free magnesium ions at a temperature between 10° C. and 30° C. 
     
     
         40 . The amplification reagent mixture of  claim 29 , further comprising between 5 mM and 50 mM free magnesium ions at a temperature between 40° C. and 70° C. 
     
     
         41 . The amplification reagent mixture of  claim 29 , wherein said amplification reagent mixture has a temperature between 40° C. and 70° C. 
     
     
         42 . The amplification reagent mixture of  claim 41 , further comprising an amplicon. 
     
     
         43 . A composition comprising a nucleic acid amplification reagent, a temperature-sensitive buffer, a pH-sensitive chelating agent, and a magnesium salt. 
     
     
         44 . The composition of  claim 43 , wherein said composition is lyophilized. 
     
     
         45 . The composition of  claim 43 , wherein said composition is provided in a cartridge, microfluidic device, fluidic device, lateral flow device, or sample preparation and transfer device. 
     
     
         46 . The composition of  claim 43 , wherein the ratio of the pH-sensitive chelating agent concentration to the magnesium salt concentration is from about 0.5 to about 2.0. 
     
     
         47 . The composition of  claim 43 , wherein said composition has a temperature between 10° C. and 30° C. 
     
     
         48 . The composition of  claim 43 , further comprising between 0 mM and 10 mM free magnesium ions at a temperature between 10° C. and 30° C. 
     
     
         49 . The composition of  claim 43 , further comprising between 5 mM and 50 mM free magnesium ions at a temperature between 40° C. and 70° C. 
     
     
         50 . The composition of  claim 43 , wherein said amplification reagent mixture has a temperature between 40° C. and 70° C. 
     
     
         51 . The composition of  claim 43 , further comprising an amplicon.