Method for controlled release of enzymatic reaction components

Abstract

Compositions and reaction kits are provided for controlling an enzymatic process. The compositions and kits include a substantially water-insoluble, water-permeable polymer and a poorly water-soluble salt of an essential ionic enzymatic reactant. The disclosed compositions and methods are particularly useful for improving the specificity and performance of PCR.

1 - 36 . (canceled) 
     
     
         37 . A composition comprising:
 at least one substantially water-insoluble, water-permeable polymer; and   at least one magnesium salt of an ionic enzymatic reactant and a counter-ion,   where the magnesium salt has a molar solubility in water less than 1×10 −3  mol/L at 25° C.   
     
     
         38 . The composition of  claim 37 , where the at least one polymer is selected from the group consisting of: butyral resin, polyvinyl acetal, poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate), polyvinyl butyrate, polyvinyl acetate and combinations and co-monomers thereof. 
     
     
         39 . The composition of  claim 37 , where the at least one polymer includes polyvinyl acetal. 
     
     
         40 . The composition of  claim 37 , where the at least one polymer includes poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate). 
     
     
         41 . The composition of  claim 37 , where the salt is selected from the group consisting of: magnesium sulfite, magnesium oxalate, magnesium fluoride, and magnesium tungstate. 
     
     
         42 . The composition of  claim 37 , where the magnesium salt is magnesium oxalate. 
     
     
         43 . The composition of  claim 37 , where the composition is in the form of a dried coating. 
     
     
         44 . The composition of  claim 37 , further comprising an enzyme requiring Mg 2+  to be enzymatically active. 
     
     
         45 . The composition of  claim 37 , where the enzyme is a thermostable enzyme selected from the group consisting of: polymerase, ligase, endonuclease, kinase, protease and combinations thereof. 
     
     
         46 . A composition for an enzymatic process comprising:
 at least one substantially water-insoluble, water-permeable polymer; and,   at least one salt of an ionic enzymatic reactant and a counter-ion, the salt having a molar solubility in water less than 1×10 −3  mol/L at 25° C.; and   at least one enzyme requiring the ionic enzymatic reactant to be enzymatically active,   
     
     
         47 . The composition of  claim 46 , where the enzyme requires Mg 2+  to be enzymatically active. 
     
     
         48 . The composition of  claim 46 , where the enzyme is a thermostable enzyme selected from the group consisting of: polymerase, ligase, endonuclease, kinase, protease and combinations thereof. 
     
     
         49 . The composition of  claim 46 , where the enzyme is a thermostable DNA polymerase. 
     
     
         50 . A reaction kit for an enzymatic process comprising:
 a first reaction vessel containing at least one substantially water-insoluble, water-permeable polymer; and   at least one magnesium salt of an ionic enzymatic reactant and a counter-ion;   where the at least one magnesium salt has a molar solubility in water less than 1×10 −3  mol/L at 25° C.   
     
     
         51 . The reaction kit of  claim 50 , where the at least one substantially water-insoluble, water-permeable polymer and the at least one salt is coated on a wall of the first reaction vessel. 
     
     
         52 . The reaction kit of  claim 51 , where the first reaction vessel is a tube, plate, slide, well or combination thereof. 
     
     
         53 . The reaction kit of  claim 50 , where the at least one substantially water-insoluble, water-permeable polymer is selected from the group consisting of: butyral resin, polyvinyl acetal, poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate), polyvinyl butyrate, polyvinyl acetate and combinations and co-monomers thereof. 
     
     
         54 . The reaction kit of  claim 50 , further comprising one or more reaction vessels, where each of the one or more reaction vessels comprises at least one reaction component. 
     
     
         55 . The reaction kit of  claim 50 , further comprising a second reaction vessel comprising at least one enzyme whose activity is dependent on the ionic enzymatic reactant. 
     
     
         56 . The reaction kit of  claim 50 , where the at least one enzyme is a thermostable enzyme selected from the group consisting of: polymerase, ligase, endonuclease, kinase, protease and combinations thereof.