Compositions and methods for the release of nucleic acid molecules from solid matrices

Abstract

The present invention relates to compositions and methods for releasing nucleic acid molecules from solid matrices. The invention further relates to compositions and methods for purifying and isolating nucleic acid molecules from biological materials such as animal tissues and plant matter. The methods of the invention can be readily adapted for rapid processing of multiple samples. Thus, the invention further provides automated methods for the purification of nucleic acid molecules from numerous samples. The invention also relates to kits for removing nucleic acid molecules from solid matrices.

1 - 58 . (canceled).  
     
     
         59 . A nucleic acid isolated by the method comprising: 
 (a) contacting the nucleic acid molecule with a solid matrix under conditions which favor adherence, attachment, association, and/or binding of the nucleic acid molecule to the solid matrix; and    (b) contacting the solid matrix containing the bound nucleic acid molecule with a releasing reagent comprising one or more alkanol amines, under conditions which favor release of the nucleic acid molecule from the solid matrix.    
     
     
         60 . (canceled).  
     
     
         61 . A recombinant host cell comprising a nucleic acid produced by a method comprising: 
 (a) contacting the nucleic acid molecule with a solid matrix under conditions which favor adherence, attachment, association, and/or binding of the nucleic acid molecule to the solid matrix;    (b) contacting the solid matrix containing the bound nucleic acid molecule with a releasing reagent comprising one or more alkanol amines, under conditions which favor release of the nucleic acid molecule from the solid matrix;    (c) amplifying the released nucleic acid molecules by PCR; and    (d) transfecting a host cell with the amplified nucleic acid molecule.    
     
     
         62 . The host cell of  claim 61 , wherein the nucleic acid molecule of (d) is contained by a vector.  
     
     
         63 . A method of making a recombinant host cell comprising introducing a nucleic acid into a host cell; wherein the nucleic acid is produced by a method comprising: 
 (a) contacting the nucleic acid molecule with a solid matrix under conditions which favor adherence, attachment, association, and/or binding of the nucleic acid molecule to the solid matrix;    (b) contacting the solid matrix containing the bound nucleic acid molecule with a releasing reagent comprising one or more alkanol amines, under conditions which favor release of the nucleic acid molecule from the solid matrix; and    (c) amplifying the released the nucleic acid molecules.    
     
     
         64 - 78 . (canceled).  
     
     
         79 . A composition comprising nucleic acid molecules, a solid matrix and a releasing reagent, wherein the releasing reagent comprises one or more alkanol amines.  
     
     
         80 . The composition of  claim 79 , wherein the solid matrix comprises a weak base, a chelating agent, and an anionic surfactant or anionic detergent.  
     
     
         81 . The composition of  claim 79 , wherein the solid matrix further comprises uric acid or a urate salt.  
     
     
         82 . The composition of  claim 79 , wherein the solid matrix is a cellulose based matrix or a micromesh synthetic plastic matrix.  
     
     
         83 . The composition of  claim 82 , wherein the solid matrix is a filter paper.  
     
     
         84 . The composition of  claim 82 , wherein the solid matrix is FTA® paper.  
     
     
         85 . The composition of  claim 79 , wherein the releasing reagent comprises more than one ethanolamine.  
     
     
         86 . The composition of  claim 79 , wherein the releasing reagent comprises an ethanolamine.  
     
     
         87 . The composition of  claim 86 , wherein the ethanolamine is mono-ethanol amine.  
     
     
         88 . The composition of  claim 86 , wherein the ethanolamine is di-ethanolamine.  
     
     
         89 . The composition of  claim 86 , wherein the ethanolamine is tri-ethanolamine.  
     
     
         90 . The composition of  claim 86 , wherein the solution is an aqueous solution.  
     
     
         91 . The composition of  claim 79 , wherein the releasing reagent has a pH from about 8.3 to about 13.  
     
     
         92 . The composition of  claim 91 , wherein the releasing reagent has a pH from about 10 to about 12.  
     
     
         93 . The composition of  claim 92 , wherein the releasing reagent has a pH of about 11.  
     
     
         94 . The composition of  claim 79 , wherein the concentration of alkanol amines is from about 0.01% to about 5% (vol./vol.).  
     
     
         95 . The composition of  claim 94 , wherein the concentration of alkanol amines is from about 0.01% to about 3% (vol./vol.).  
     
     
         96 . The composition of  claim 95 , wherein the concentration of alkanol amines is from about 0.01% to about 1% (vol./vol.).  
     
     
         97 . The composition of  claim 96 , wherein the concentration of alkanol amines is from about 0.1% to about 1% (vol./vol.).  
     
     
         98 . The composition of  claim 79 , wherein the nucleic acid molecules comprise DNA.  
     
     
         99 . The composition of  claim 79 , wherein the nucleic acid molecules comprise a vector.  
     
     
         100 . The composition of  claim 99 , wherein the vector comprises a plasmid or an artificial chromosome.  
     
     
         101 . The composition of  claim 79 , wherein the nucleic acid molecules are from a cell or a virus.  
     
     
         102 . A kit for removing nucleic acid molecules from a solid matrix, the kit comprising (1) a releasing reagent comprising one or more alkanol amines and (2) one or more components selected from the group consisting of: 
 (a) a solid matrix;    (b) an apparatus for applying samples to a solid matrix;    (c) an apparatus cutting a solid matrix into sections which contain samples; and    (d) a washing solution.    
     
     
         103 . The kit of  claim 102 , wherein the solid matrix comprises a weak base, a chelating agent, and an anionic surfactant or anionic detergent.  
     
     
         104 . The kit of  claim 102 , wherein the solid matrix further comprises uric acid or a urate salt.  
     
     
         105 . The kit of  claim 102 , wherein the solid matrix is a cellulose based matrix or a micromesh synthetic plastic matrix.  
     
     
         106 . The kit of  claim 105 , wherein the solid matrix is a filter paper.  
     
     
         107 . The kit of  claim 105 , wherein the solid matrix is FTA® paper.  
     
     
         108 . The kit of  claim 102 , wherein the releasing reagent comprises more than one ethanolamine.  
     
     
         109 . The kit of  claim 102 , wherein the releasing reagent comprises an ethanolamine.  
     
     
         110 . The kit of  claim 109 , wherein the ethanolamine is monoethanolamine.  
     
     
         111 . The kit of  claim 109 , wherein the ethanolamine is diethanolamine.  
     
     
         112 . The kit of  claim 109 , wherein the ethanolamine is triethanolamine.  
     
     
         113 . The kit of  claim 102 , wherein the solution is an aqueous solution.  
     
     
         114 . The kit of  claim 102 , wherein the releasing reagent has a pH from about 8.3 to about 13.  
     
     
         115 . The kit of  claim 114 , wherein the releasing reagent has a pH from about 10 to about 12.  
     
     
         116 . The kit of  claim 115 , wherein the releasing reagent has a pH of about 11.  
     
     
         117 . The kit of  claim 102 , wherein the concentration of alkanol amines is from about 0.01% to about 5% (vol./vol.).  
     
     
         118 . The kit of  claim 117 , wherein the concentration of alkanol amines is from about 0.01% to about 3% (vol./vol.).  
     
     
         119 . The kit of  claim 118 , wherein the concentration of alkanol amines is from about 0.01% to about 1% (vol./vol.).  
     
     
         120 . The kit of  claim 119 , wherein the concentration of alkanol amines is from about 0.1% to about 1% (vol./vol.).  
     
     
         121 . The kit of  claim 102 , wherein the nucleic acid molecules comprise a vector.  
     
     
         122 . The kit of  claim 121 , wherein the vector comprises a plasmid or an artificial chromosome.  
     
     
         123 . The kit of  claim 102 , wherein the nucleic acid molecules are from a cell or a virus.  
     
     
         124 . The kit of  claim 102 , wherein the apparatus for applying samples to a solid matrix comprises a micropipette.  
     
     
         125 . The kit of  claim 102 , wherein the apparatus for applying samples to a solid matrix is capable of applying multiple samples to the solid matrix at one time.  
     
     
         126 . The kit of  claim 102 , wherein the apparatus for applying samples to a solid matrix results in the samples being crushed into the surface of the solid matrix.  
     
     
         127 . The kit of  claim 102 , wherein the apparatus cutting the solid matrix into pieces which are circular, square, rectangular, or irregular in shape.  
     
     
         128 . The kit of  claim 102 , wherein the washing solution comprises 10 mM Tris HCl, 1 mM EDTA (pH 7.3).  
     
     
         129 . The kit of  claim 102 , wherein the washing solution further comprises a detergent.