Systems and methods for the rapid isolation of nucleic acids

Abstract

The present invention contemplates a system for rapidly isolating nucleic acids. The system comprises an insoluble silica matrix and a buffered aqueous salt solution containing salt at a concentration of at least 3 molar and a buffering agent at a concentration sufficient to provide a buffering capacity corresponding to that which either tris(hydroxymethyl)aminomethane or phosphate ion at a concentration of 0.1 to 1 molar would provide in the solution. Methods of using the system are also contemplated.

What is claimed is:  
     
         1 . A system, in kit form, for isolating plasmid DNA from an aqueous sample, which system comprises, in separate containers, particulate glass and a buffered aqueous salt solution having a pH value in the range of 7 to 8, said solution containing; 
 a) a salt at a concentration of at least 3 molar, and    b) a buffering agent at a concentration sufficient to provide a buffering capacity corresponding to that which 0.1 to 1 molar tris(hydroxymethyl)aminomethane or 0.1 to 1 molar phosphate ion would provide in said solution.    
     
     
         2 . The system of  claim 1  wherein said buffered salt solution is substantially free of cyclohexanediamine tetraacetate.  
     
     
         3 . The system of  claim 1  further containing a sieve at least about 1 inch in diameter and having a mesh size in the range of 90 to 350.  
     
     
         4 . The system of  claim 1  further containing, in a separate package, a sample of control plasmid DNA.  
     
     
         5 . The system of  claim 4  wherein said control plasmid DNA is present in a viable host cell capable of supporting replication of said control plasmid DNA.  
     
     
         6 . The system of  claim 1  further containing, in a separate package, a unit dose of a dry-concentrate of a culture medium capable of supporting growth of cells containing said plasmid DNA.  
     
     
         7 . The system of  claim 6  wherein said medium is in tablet or capsular form.  
     
     
         8 . The system of  claim 1  wherein said buffered aqueous salt solution has a pH value in the range of 7.2 to 7.8.  
     
     
         9 . The system of  claim 1  wherein said salt is selected from the group consisting of NaI, NaBr, NaCl, KI, KBr, CsCl, GNHCl and GNSCN.  
     
     
         10 . The system of  claim 1  wherein said salt concentration is in the range of 4 to 6 molar.  
     
     
         11 . The system of  claim 1  wherein said particulate glass has a sedimentation rate through still water at unit gravity in the range of about 0.001 to about 1.0 cm/min.  
     
     
         12 . A system, in kit form, for isolating plasmid DNA from a sample containing RNA and said DNA, which system comprises, in separate containers; 
 a) particulate glass; and    b) a buffered aqueous salt solution having a pH value in the range of 7.2-7.8, said solution consisting essentially of: 
 i) 2 M NaI,  
 ii) 2.6 M KBr, and  
 iii) 0.66 M tris(hydroxymethyl)aminomethane.  
   
     
     
         13 . The system of  claim 12  further containing, in unit dose form, a dry-concentrate of a culture medium capable of supporting growth of cells containing said plasmid DNA.  
     
     
         14 . A system, in kit form, for isolating nucleic acid molecules, which system comprises a composition comprising particulate glass having a sedimentation rate through still water at unit gravity in the range of about 0.001 to about 1.0 cm/min.  
     
     
         15 . A system, in kit form, for isolating DNA from an aqueous sample, which system comprises, in separate containers, particulate glass and a dry buffered salt admixture which upon dissolution in a predetermined amount of distilled water provides a solution having a pH value in the range of 7 to 8, said buffered salt admixture containing: 
 a) a salt in an amount sufficient to provide a concentration of at least 3 molar upon said dissolution, and    b) a buffering agent at a concentration sufficient to provide a buffering capacity corresponding to that provided by 0.1 to 1 molar aqueous tris(hydroxymethyl)aminomethane or 0.1 to 1 molar aqueous phosphate ion.    
     
     
         16 . A method for isolating plasmid DNA from a sample containing RNA and said DNA, which method comprises: 
 a) forming a binding reaction admixture by admixing said sample with an insoluble silica matrix and a buffered aqueous salt solution having a pH value in the range of 7 to 8, said solution containing i) a salt at a concentration of at least 3 molar, and ii) a buffering agent at a concentration sufficient to provide a buffering capacity corresponding to that which 0.1 to 1 molar tris(hydroxymethyl)aminomethane or 0.1 to 1 molar phosphate ion would provide in said solution;    b) maintaining said binding reaction admixture for a time period sufficient for said DNA to bind to said matrix to form an insoluble DNA-matrix complex and a remaining admixture;    c) separating said remaining admixture and said complex to form an isolated complex; and    d) recovering said DNA from said isolated complex to form isolated plasmid DNA.    
     
     
         17 . A method for isolating DNA from an agarose gel sample containing said DNA, which method comprises: 
 a) forming a gel-dissolving reaction admixture by admixing said sample with a buffered aqueous chaotropic salt solution having a pH value in the range of 7 to 8, said solution containing i) a chaotropic salt at a concentration of at least 3 molar, and ii) a buffering agent at a concentration sufficient to provide a buffering capacity corresponding to that which 0.1 to 1 molar tris(hydroxymethyl)aminomethane or 0.1 to 1 molar phosphate ion would provide in said solution;    b) maintaining said gel-dissolving reaction admixture at a temperature of about 45 to about 65 degrees C. for a time period sufficient for said gel sample to dissolve to form a dissolved sample;    c) admixing said dissolved sample with an insoluble silica matrix to form a binding reaction admixture;    d) maintaining said binding reaction admixture for a time period sufficient for said DNA present in said sample to bind to said matrix to form a solution containing dissolved agarose and an insoluble DNA-matrix complex;    e) separating said complex from said dissolved agarose to form an isolated complex; and    f) recovering said DNA from said isolated complex to form isolated DNA.    
     
     
         18 . A dry-concentrate culture medium composition in unit dose comprising an amount of cell culture medium in dry-concentrate form sufficient to prepare a preselected amount of culture medium, said dry medium packaged in unit dose form.  
     
     
         19 . The composition of  claim 18  wherein said unit dose packaging is in the form of a capsule containing said dry culture medium.  
     
     
         20 . The composition of  claim 19  wherein said dry culture medium is LB-broth.  
     
     
         21 . The composition of  claim 18  wherein said unit dose packaging is dissolvable.