Process for performing polynucleotide separations

Abstract

The invention recognizes the deleterious effects of trace, and even undetectable amounts of multivalent cations on the separation of mixtures of polynucleotides, especially double stranded polynucleotides, and provides an improved method for separating such mixtures on wide pore, non-polar separation media by eliminating multivalent cations from the all aspects of the separation process. This is accomplished by using components in the separation process which are materials which do not release metal cations. In addition, the use of cation capture resins and other methods to remove residual traces of multivalent cations from eluting solvents, sample solutions, separation media, and system components is described. It is also important to remove any traces or organic contaminants from solvents solutions and system parts. Taking similar steps to remove residual traces of multivalent cations and organic impurities from the separation process, the invention may also be used in a batch process to separate mixtures of polynucleotide fragments.

What is claimed is:  
     
         1 . A method for separating a mixture of polynucleotide fragments comprising 
 a) applying a solution of said fragments and counterion reagent to a column containing, separation media having a non-polar surface, wherein said separation media have a pore size greater than 30 Angstroms and an average diameter of 1-100 microns;    b) eluting said fragments with a gradient eluting solvent of increasing organic component concentration containing a counterion agent;    wherein surfaces which are contacted by the solution of the fragments and the eluting solvent are materials which do not trap or release multivalent metal cations therefrom.    
     
     
         2 . A method of  claim 1  wherein the solution of said fragments and the eluting solvent are contacted with a multivalent cation capture resin to remove any multivalent cations therein before entering the column.  
     
     
         3 . A method of  claim 2  wherein said separation media have been treated to remove residual traces of multivalent cations from the surfaces thereof.  
     
     
         4 . A method of  claim 3  wherein the solution or said fragments and eluting solvent have been contacted with a multivalent cation capture resin before entering the column.  
     
     
         5 . A method of  claim 4  wherein the polynucleotide fragments are double stranded or single stranded.  
     
     
         6 . A method of  claim 5  wherein the fragments having more than 5 base pairs are separated on the basis of size or polarity.  
     
     
         7 . A method of  claim 4  wherein said separation media are organic polymer.  
     
     
         8 . A method of  claim 4  wherein said separation media have inorganic substrates selected from the group consisting of inorganic substrates, silica, zirconia, and alumina.  
     
     
         9 . A method of  claim 8  wherein the non-polar surface is an organic polymer supported on the inorganic substrate.  
     
     
         10 . A method of  claim 8  wherein the non-polar surface includes long chain hydrocarbon groups having from 8 to 24 carbons bound to the inorganic substrate.  
     
     
         11 . A method of  claim 10  wherein residual polar groups of the inorganic substrate have been end capped with trimethylsilyl chloride or hexamethyldisilazane.  
     
     
         12 . A method of  claim 4 , wherein the surfaces contacted by the solution of polynucleotide fragments and eluting solvent are titanium, coated stainless steel, organic polymer or combinations thereof.  
     
     
         13 . A method of  claim 12  wherein traces of residual multivalent metal cations have been removed from said surfaces by treating said surfaces with a solution comprising aqueous acid and chelating agent.  
     
     
         14 . A method of  claim 12  wherein organic contaminants have been removed from said surfaces.  
     
     
         15 . A method of  claim 1  wherein said solution of polynucleotide mixture and eluting solvent contain a chelating agent, whereby any trace of multivalent metal cations in the solutions are captured.  
     
     
         16 . A method of  claim 4  wherein said solution of polynucleotide mixture and eluting solvent contain a chelating agent, whereby any trace of multivalent metal cations in the solutions are captured.  
     
     
         17 . A method of  claim 4  wherein the eluting solvent has been treated to remove oxygen therefrom.  
     
     
         18 . A method of  claim 4  wherein said method for separating said mixture of polynucleotides comprises Matched Ion Polynucleotide Chromatography.  
     
     
         19 . A batch process for separating polynucleotide fragments having a selected size from a mixture of polynucleotide fragments including fragments of said selected size comprising 
 a) applying a solution of said polynucleotide fragments and a counterion agent to non-polar separation media having a non-polar surface, wherein said separation media have a pore size greater than 30 Angstroms and an average diameter of 1-100 microns;    b) contacting the separation media with a first eluting solvent and counterion agent, the first eluting solvent having a concentration of organic component sufficient to release from the separation media all polynucleotide fragments having a size smaller than the selected size and removing the first eluting solvent from the separation media; and    c) contacting the separation media with a second eluting solvent having a concentration of organic component sufficient to release from the separation media the polynucleotide fragments having the selected size and removing the second eluting solvent from the separation media;    wherein surfaces which are contacted by the solution of polynucleotide fragments and the eluting solvent are material which does not trap or release multivalent metal cations therefrom.    
     
     
         20 . A batch process of  claim 19  wherein the separation media are rinsed with fresh first eluting solvent following step b) to remove residual released polynucleotide fragments therefrom.  
     
     
         21 . A batch process of  claim 19  wherein the separation media are rinsed with fresh second eluting solvent following step c) to remove residual released polynucleotide fragments of selected size therefrom.  
     
     
         22 . A batch process of  claim 19  wherein the solution of polynucleotide mixture and eluting solvent have been contacted with a multivalent cation capture resin before contacting the separation media.  
     
     
         23 . A batch process of  claim 19  wherein the polynucleotide mixture is double stranded or single stranded.  
     
     
         24 . A batch process of  claim 22  wherein said separation media have been treated to remove residual traces of multivalent cations therefrom.  
     
     
         25 . A batch process of  claim 24  wherein the solution of polynucleotide mixture and eluting solvent have been contacted with a multivalent cation capture resin before contacting the separation media.  
     
     
         26 . A batch process of  claim 25  wherein the separation media are contained in a column, a web, a membrane, or container.  
     
     
         27 . A batch process of  claim 26  wherein said separation media are organic polymer or inorganic substrates selected from the group consisting of inorganic substrates, silica, zirconium, and alumina.  
     
     
         28 . A batch process of  claim 27  wherein the non-polar surface is an organic polymer supported on the inorganic substrate.  
     
     
         29 . A batch process of  claim 27  wherein the non-polar surface includes long chain hydrocarbons having from 8-24 carbons bonded the inorganic substrate.  
     
     
         30 . A batch process of  claim 29  wherein any residual polar groups of the inorganic substrate have been end capped with trimethylsilyl chloride or hexamethyldisilazane.  
     
     
         31 . A batch process of  claim 26 , wherein the surfaces contacted by the solution of polynucleotide fragments and eluting solvent are comprised of material selected from the group consisting of titanium, coated stainless steel, and organic polymer, or combinations thereof.  
     
     
         32 . A batch process of  claim 31  wherein traces of residual multivalent metal cations have been removed from said surfaces by treating said surfaces with a solution comprising aqueous acid and chelating agent.  
     
     
         33 . A batch process of  claim 31  wherein organic contaminants have been removed from said surfaces.  
     
     
         34 . A batch process of  claim 25  wherein said solution of polynucleotide mixture and eluting solvent contain a chelating agent whereby any trace of multivalent metal cations in the solution are captured.  
     
     
         35 . A batch process of  claim 19  wherein the eluting solvent has been treated to remove oxygen therefrom.