Isolation of nucleic acid

Abstract

A method for isolating a nucleic acid comprises: binding the nucleic acid to a solid phase at a first pH in the presence of a binding buffer, washing the bound nucleic acid with a wash solution, and eluting the nucleic acid from the solid phase at a second pH which is higher than the first pH. The wash solution comprises a buffer with a buffering range that encompasses a pH that is higher than the first pH, and the wash solution is at a pH that is within a buffering range of the binding buffer but lower than the buffering range of the buffer of the wash solution. Solutions, compositions, and kits for use in the methods are described.

1 . A method for isolating a nucleic acid, which comprises:
 (i) binding the nucleic acid to a solid phase at a first pH in the presence of a binding buffer;   (ii) washing the bound nucleic acid with a wash solution; and   (iii) eluting the nucleic acid from the solid phase at a second pH which is higher than the first pH;   
       wherein the wash solution comprises a buffer with a buffering range that encompasses a pH that is higher than the first pH, and the wash solution is at a pH that is within a buffering range of the binding buffer but lower than the buffering range of the buffer of the wash solution; and wherein the pH of the wash solution is pH 6.0 or less, and wherein the nucleic acid is eluted from the solid phase at a temperature above ambient temperature. 
     
     
         2 . The method of  claim 1 , wherein the buffering range of the wash buffer is higher than the first pH. 
     
     
         3 . The method of  claim 1 , wherein the second pH is within the buffering range of the wash buffer. 
     
     
         4 . The method of  claim 1 , wherein the buffering range of the wash buffer is higher than pH 5.0. 
     
     
         5 . The method of  claim 1 , wherein the first pH is in the range pH 3-6 or pH 3-5, the second pH is in the range pH 6.5-10 or pH 7-9, or the pH of the wash solution is from pH 3.0 to pH 6.0. 
     
     
         6 .- 7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the first pH is within the buffering range of the binding buffer. 
     
     
         9 . The method of  claim 1 , wherein a lower end of the buffering range of the binding buffer is at pH 3.0 or higher. 
     
     
         10 . The method of  claim 1 , wherein the method is carried out in the absence of a chaotropic agent and/or an organic solvent. 
     
     
         11 . The method of  claim 1 , wherein binding of the nucleic acid to the solid phase is carried out in the presence of a kosmotropic agent, wherein optionally the binding buffer comprises the kosmotropic agent. 
     
     
         12 . The method of  claim 11 , wherein the kosmotropic agent is ammonium sulphate. 
     
     
         13 . The method of  claim 1 , wherein the solid phase comprises a negatively ionisable group with a pKa between the first and second pH. 
     
     
         14 . The method of  claim 1 , wherein the solid phase comprises an inorganic oxide, wherein optionally the inorganic oxide is silica. 
     
     
         15 . A method for isolating a nucleic acid from a cell, which comprises lysing the cell to release the nucleic acid from the cell, and isolating the released nucleic acid using a method according to  claim 1 . 
     
     
         16 . The method of  claim 15 , wherein lysis is carried out using the binding buffer. 
     
     
         17 .- 18 . (canceled) 
     
     
         19 . The method of  claim 1 , wherein the nucleic acid is eluted from the solid phase in the presence of an elution buffer, wherein the second pH is within a buffering range of the elution buffer, and wherein optionally the buffering range of the elution buffer overlaps with or is encompassed by the buffering range of the wash buffer. 
     
     
         20 .- 29 . (canceled) 
     
     
         30 . A kit for isolation of a nucleic acid, which comprises:
 a) i) a binding buffer for binding the nucleic acid to a solid phase at a first pH, wherein optionally the binding buffer comprises a kosmotropic agent;
 ii) a wash solution that comprises a buffer with a buffering range that encompasses a pH that is higher than the first pH, wherein the wash solution is at a pH that is within a buffering range of the binding buffer but lower than the buffering range of the wash buffer; and optionally 
 iii) a solution for eluting the nucleic acid from the solid phase, wherein the solution is at a second pH that is higher than the first pH or 
   b) i) a binding buffer for binding the nucleic acid to a solid phase at a first pH, wherein optionally the binding buffer comprises a kosmotropic agent;
 ii) a composition in dry form that when dissolved in a liquid provides a wash solution that comprises a buffer with a buffering range that encompasses a pH that is higher than the first pH, wherein the wash solution is at a pH that is within a buffering range of the binding buffer but lower than the buffering range of the buffer of the wash solution; and optionally 
 iii) a composition in dry form that when dissolved in a liquid provides a solution for eluting the nucleic acid from the solid phase, wherein the solution is at a second pH that is higher than the first pH; 
 wherein the kit does not comprise a chaotropic agent and/or an organic solvent. 
   
     
     
         31 .- 35 . (canceled) 
     
     
         36 . The kit of  claim 30 , which further comprises:
 i) a solid phase to which the nucleic acid is able to bind in the presence of the binding buffer at the first pH, and from which the nucleic acid can be eluted at the second pH; or   ii) a protease, wherein optionally the protease is in lyophilised form, separate from the binding buffer.   
     
     
         37 . The kit of  claim 36 , wherein the solid phase comprises a negatively ionisable group with a pKa between a first pH at which the nucleic acid is able to bind to the solid phase, and a second pH at which the nucleic acid can be eluted from the solid phase. 
     
     
         38 . The kit of  claim 36 , wherein the solid phase comprises an inorganic oxide, wherein optionally the inorganic oxide is silica. 
     
     
         39 .- 40 . (canceled) 
     
     
         41 . The method of  claim 1 , wherein the nucleic acid is eluted from the solid phase at a temperature of 50-90° C.