Compositions and methods for using a solid support to purify DNA

Abstract

Reagents, methods and kits for the purification of DNA from biological materials are provided.

1 . A formulation for isolating and purifying DNA comprising: 
 a lithium salt at a concentration of at least about 1 M,    at least one surfactant, and    a buffer.    
     
     
         2 . The formulation of  claim 1 , further comprising a chelating agent.  
     
     
         3 . The formulation of  claim 1 , wherein the chelating agent is EDTA or citrate.  
     
     
         4 . The formulation of  claim 1 , wherein the formulation lacks a chaotrope and/or a strong chaotropic substance.  
     
     
         5 . The formulation of  claim 1 , wherein the lithium salt is lithium chloride.  
     
     
         6 . The formulation of  claim 1 , wherein the lithium salts is at a concentration of 2-10 M.  
     
     
         7 . The formulation of  claim 1 , wherein the formulation has pH of above about 7.  
     
     
         8 . The formulation of  claim 1 , wherein the solution has a pH between about 7 and about  
     
     
         9 . The formulation of  claim 1 , wherein the surfactant is present at a concentration of about 10-40% v/v.  
     
     
         10 . The formulation of  claim 1 , wherein at least one surfactant is a detergent.  
     
     
         11 . The formulation of  claim 10 , wherein the detergent is an anionic, cationic, zwitterionic or non-ionic detergent.  
     
     
         12 . The formulation of  claim 1 , wherein the surfactant is diethyl glycol monoethyl ether (DGME).  
     
     
         13 . The formulation of  claim 12 , wherein the DGME is present at a concentration of about 5% to about 35% v/v.  
     
     
         14 . The formulation of  claim 10 , wherein the detergent is a mixture of anionic and non-ionic detergents.  
     
     
         15 . The formulation of  claim 11 , wherein the anionic detergent is SDS.  
     
     
         16 . The formulation of  claim 14 , wherein the SDS is present at a concentration of between 0.05-0.2% v/v.  
     
     
         17 . The formulation of  claim 12 , wherein the detergent is Triton-X.  
     
     
         18 . The formulation of  claim 17 , wherein the detergent is present at a concentration of between 0.05-5.0% v/v.  
     
     
         19 . A method for isolating substantially pure and undegraded DNA from biological material, comprising the steps of: 
 (a) contacting the biological material with a DNA Lysing Solution to form a mixture, wherein the DNA Lysing Solution is buffered at pH of greater than 7, and wherein the DNA Lysing Solution comprises a lithium salt and at least one surfactant;    (b) contacting the mixture with a DNA Spiking Solution;    (c) contacting the mixture to a solid support such that DNA present in the biological material binds to the solid support;    (d) washing the sold support with a Wash Solution; and    (e) eluting the DNA with a DNA Elution Solution.    
     
     
         20 . The method of  claim 19 , wherein the alcohol is isopropanol, ethanol or methanol.  
     
     
         21 . The method of  claim 19 , wherein the alcohol may be a mixture of alcohols.  
     
     
         22 . The method of  claim 19 , wherein the alcohol is 100% isopropanol.  
     
     
         23 . The method of  claim 19 , wherein the DNA Spiking Solution contains an alkali-metal salt greater than 1M.  
     
     
         24 . The method of  claim 19 , wherein the biological material is a cervical cell sample.  
     
     
         25 . The method of method of  claim 19 , wherein the biological material is whole blood.  
     
     
         26 . The method of  claim 19 , wherein the biological material is a cultured cell, fixed cell, and/or tissue sample.  
     
     
         27 . The method of  claim 19 , wherein the Wash Solution is buffered at a pH of greater than about 7.  
     
     
         28 . The method of  claim 19 , wherein the Lysing and/or Wash Solution lack a chaotrope and/or a strong chaotropic substance.  
     
     
         29 . The method of  claim 19 , wherein the DNA Lysing Solution further comprises a chelating agent.  
     
     
         30 . The method of  claim 19 , wherein the solid support comprises components of silica, cellulose, cellulose acetate, nitrocellulose, nylon, polyester, polyethersulfone, polyolefin, or polyvinylidene fluoride, or combinations thereof.  
     
     
         31 . The method of  claim 19 , wherein the solid support is pre-treated with RNase solution prior to contacting the biological material with the solid support.  
     
     
         32 . The method of  claim 19 , wherein the lithium salt of the Lysing Solution is lithium chloride or lithium bromide.  
     
     
         33 . The method of  claim 19 , wherein the lithium salt of the Lysing Solution is present at a concentration greater than about 1 M.  
     
     
         34 . The method of  claim 19 , wherein the lithium salt of the Lysing Solution is present at a concentration of between 2 M and 8 M.  
     
     
         35 . The method of  claim 19 , wherein the Lysing Solution further comprises a chelating agent.  
     
     
         36 . The method of  claim 35 , wherein the chelating agent is EDTA or citrate.  
     
     
         37 . The method of  claim 19 , wherein the DNA Lysing Solution comprises a surfactant at a concentration of between about 25-35% v/v.  
     
     
         38 . The method of  claim 19 , wherein the surfactant is a detergent.  
     
     
         39 . The method of  claim 38 , wherein the detergent is a non-ionic detergent.  
     
     
         40 . The method of  claim 39 , wherein the non-ionic detergent is a Tween, Triton, Nonidet, Igepal or Tergitol.  
     
     
         41 . The method of  claim 38 , wherein the detergent is an anionic detergent.  
     
     
         42 . The method of  claim 41 , wherein the anionic detergent is SDS (sodium dodecyl sulfate) or N-lauroyl sarcosine.  
     
     
         43 . The method of  claim 19 , wherein the surfactant is a mixture of detergents, or a mixture of detergents with a solubilizing surfactant.  
     
     
         44 . The method of  claim 43 , wherein the solubilizing surfactant is DGME.  
     
     
         45 . A method for purifying substantially pure and undegraded DNA from biological material, comprising the steps of: 
 (a) contacting a biological material containing DNA with a solid support pre-treated with a DNA Lysing Solution, wherein the DNA Lysing Solution is buffered at a pH of greater than about 7, and wherein the DNA Lysing Solution comprises a surfactant and a lithium salt;    (b) adding a DNA Spiking Solution to the biological material;    (c) contacting the biological material to the solid support in order to release nucleic acids comprising substantially undegraded DNA and non-nucleic acid biological matter, wherein the nucleic acids comprising substantially undegraded DNA bind to the solid support;    (d) washing the solid support with a Wash Solution to remove biological materials other than bound nucleic acids comprising undegraded DNA; and    (e) eluting the bound undegraded DNA from the solid support with a DNA Elution Solution.    
     
     
         46 . A direct lysis method for purifying substantially pure and undegraded DNA from biological material without using a red blood cell lysis step, comprising: 
 (a) contacting a biological material containing DNA, with a first DNA Lysing Solution, wherein the first DNA Lysing Solution is buffered at a pH of greater than about 7 and comprises a surfactant at a concentration of between about 5-15% v/v and a DNA-complexing salt at a concentration of greater than 1M,    (b) contracting the biological material with a second DNA Lysing Solution, wherein the second DNA Lysing Solution comprises a surfactant and a DNA-complexing salt greater than 1M; (c) contacting the mixture with a DNA Spiking Solution;    (d) contacting the mixture with a solid support, wherein nucleic acids comprising substantially undegraded DNA from the biological material bind to the solid support;    (e) washing the solid support with a DNA Wash Solution to remove biological materials other than bound nucleic acids comprising substantially undegraded DNA, the DNA Wash Solution comprising a DNA-complexing salt and an alcohol; and    (f) preferentially eluting the bound substantially undegraded DNA from the solid support with a DNA Elution Solution.    
     
     
         47 . The method of  claim 46 , wherein the second DNA Lysing Solution further comprises a chelating agent.  
     
     
         48 . A method for purifying substantially pure and undegraded DNA from biological material comprising: 
 (a) contacting a biological material containing DNA with DNA Lysing Solution buffered at a pH of greater than about 7, wherein the DNA Lysing Solution comprises a surfactant and an DNA-complexing salt at a concentration greater than 1 M;    (b) contacting the biological material to a solid support, wherein nucleic acids comprising substantially undegraded DNA bind to the solid support;    (c) contacting the solid support with a DNA Spiking Solution;    (d) washing the solid support with a DNA Wash Solution to remove biological materials other than bound nucleic acids comprising undegraded DNA, the DNA Wash Solution comprising a DNA-complexing salt and an alcohol; and    (e) preferentially eluting bound substantially undegraded DNA from the solid support with a DNA Elution Solution.    
     
     
         49 . A method for purifying substantially pure and undegraded DNA fixed cervical cell samples, comprising the steps of 
 (a) contacting a biological material containing DNA with DNA Lysing Solution buffered at a pH of greater than about 7, wherein the DNA Lysing Solution containing a surfactant at a concentration of between about 25-35% V/v, a chelating agent, and a DNA-complexing salt of greater than 1 M;    (b) adding to the mixture, an optional DNA Spiking Solution containing alcohol;    (c) contacting the biological material to the solid support in order to release nucleic acids comprising substantially undegraded DNA and non-nucleic acid biological matter causing nucleic acids comprising substantially undegraded DNA to bind to the solid support;    (d) washing the solid support with a DNA Wash Solution to remove biological materials other than bound nucleic acids comprising undegraded DNA; and    (e) preferentially eluting the bound undegraded DNA from the solid support with a DNA Elution Solution in order to obtain substantially pure and undegraded DNA.