Methods for nucleic acid capture

Abstract

Solutions, reagents, and methods for nucleic acid purification. In certain aspects, cationic surfactant and, optionally, an anionic surfactant solutions are provided which can be used for phase separation and capture of nucleic acids, such as plasmid or genomic DNA, to a solid phase carrier, such as a mineral matrix.

1 . A method of isolating DNA, comprising:
 (a) obtaining a sample comprising DNA;   (b) capturing the DNA to a mineral matrix with a phase separation solution comprising domiphen bromide (DB) and a salt selected from the group consisting of lithium salt, sodium salt, potassium salt, magnesium salt, or calcium salt, and combinations thereof;   (c) treating the captured DNA with a salt solution, thereby increasing the retention of the captured DNA;   (d) washing the mineral matrix and captured DNA with a wash solution; and   (e) eluting the DNA from the mineral matrix, thereby isolating the DNA.   
     
     
         2 . The method of  claim 1 , wherein the treatment solution comprises a lithium salt, sodium salt, or potassium salt, and the wash solution is an organic wash solution. 
     
     
         3 . The method of  claim 1 , wherein the wash solution comprises an alcohol. 
     
     
         4 . The method of  claim 3 , wherein the wash solution comprises at least 60% ethanol or isopropanol. 
     
     
         5 . The method of  claim 1 , wherein (b) capturing DNA to a mineral matrix is in the presence of 0.05% to 2% DB. 
     
     
         6 . The method of  claim 1 , further defined as a method of selectively isolating genomic DNA, wherein the phase separation solution comprises a Sodium salt, lithium salt, or potassium salt. 
     
     
         7 . The method of  claim 6 , wherein (b) capturing genomic DNA to a mineral matrix is in the presence of 0.05% to 1% DB and 0.05 M to 1.0 M lithjum chloride, sodium chloride, potassium chloride, lithium acetate, sodium acetate, or potassium acetate. 
     
     
         8 . The method of  claim 1 , further defined as a method of selectively isolating plasmid DNA, wherein the phase separation solution comprises a Lithium salt, sodium salt, or potassium salt. 
     
     
         9 . The method of  claim 8 , wherein (b) capturing DNA to a mineral matrix is in the presence of 0.05% to 1% DB and 0.05 M to 1.0 M lithium chloride, sodium chloride, potassium chloride, lithium acetate, sodium acetate, or potassium acetate. 
     
     
         10 . The method of  claim 1 , wherein the obtaining a sample comprising plasmid DNA comprises obtaining a bacterial cell lysate by alkaline lysis. 
     
     
         11 . The method of  claim 10 , wherein obtaining the bacterial cell lysate comprises:
 (i) lysing cells comprising nucleic acid including plasmid DNA with a basic solution thereby generating a lysate;   (ii) neutralizing the lysate with an acidic solution thereby precipitating a genomic DNA fraction and proteins; and   (iii) clearing the precipitate.   
     
     
         12 . The method of  claim 1 , wherein the isolated DNA is essentially free of RNA, endotoxin and/or PCR inhibitors. 
     
     
         13 . A method for capture of nucleic acid on a mineral matrix for purification comprising:
 (a) contacting a nucleic acid-containing sample with a phase separation solution comprising a phase separation reagent of Formula I:   
       
         
           
           
               
               
           
         
         wherein:
 X 1  is nitrogen, phosphorus, N-heteroaryl (C≦18)  or substituted N-heteroaryl (C≦18) , provided that when X 1  is N-heteroaryl (C≦18)  or substituted N-heteroaryl (C≦18) , then R 2 , R 3 , and R 4  are absent and when R 2 , R 3 , and R 4  are absent, then X 1  is N-heteroaryl (C≦18)  or substituted N-heteroaryl (C≦18) ; 
 R 1  is alkyl (C≦30) , alkenyl (C≦30) , alkynyl (C≦30) , aryl (C≦30) , aralkyl (C≦30) , heteroaryl (C≦30) , heteroaralkyl (C≦30) , a substituted version of any of these groups or —Y 1 —Z—Y 2 ;
 Y 1  is alkandiyl (C≦6) , alkendiyl (c≦6) , alkyndiyl (C≦6) , arenediyl (C≦6) , alkoxydiyl (C≦6) , or a substituted version of any of these groups; 
 Z is —O—, —NH—, —S—, —C(O)O—, —OC(O)—, —NHC(O)—; or —C(O)NH—; 
 Y 2  is alkyl (C≦30) , alkenyl (C≦30) , alkynyl (C≦30) , aryl (C≦30) , aralkyl (C≦30) , heteroaryl (C≦30) , heteroaralkyl (C≦30) , or a substituted version of any of these groups; 
 
 R 2 , R 3 , and R 4  are each independently alkyl (C≦30) , alkenyl (C≦30) , alkynyl (C≦30) , aryl (C≦30) , aralkyl (C≦30) , heteroaryl (C≦30) , heteroaralkyl (C≦30) , a substituted version of any of these groups or —Y 3 —Z—Y 4 ;
 Y 3  is alkandiyl (C≦6) , alkendiyl (C≦6) , alkyndiyl (C≦6) , arenediyl (C≦6) , or a substituted version of any of these groups;
 Z is —O—, —NH—, —S—, —C(O)O—, —OC(O)—, —NHC(O)—; or —C(O)NH—; 
 
 Y 4  is alkyl (C≦30) , alkenyl (C≦30) , alkynyl (C≦30) , aryl (C≦30) , aralkyl (C≦30) , heteroaryl (C≦30) , heteroaralkyl (C≦30) , or a substituted version of any of these groups; 
 
 A is bromide, chloride, iodide, phosphate, sulfate, acetate, formate, propionate, oxalate, or succinate; 
 
         provided that when R 2 , R 3  and R 4  are alkyl (C≦30) , alkenyl (C≦30) , alkynyl (C≦30) , aryl (C≦30) , aralkyl (C≦30) , heteroaryl (C≦30) , heteroaralkyl (C≦30) , or a halo substituted version of any of these groups and X 1  is nitrogen or phosphorus, then R 1  is —Y 1 —Z—Y 2 , to provide a prepared sample of phase separated nucleic acid; and 
         (b) capturing the phase separated nucleic acid from the prepared sample to the mineral matrix to provide captured nucleic acid. 
       
     
     
         14 . The method of  claim 13 , further comprising:
 (c) treating the captured nucleic acid with a salt solution, thereby increasing the retention of the captured nucleic acid;   (d) washing the captured nucleic acid with an organic wash solution, thereby purifying the captured nucleic acid; and   (e) eluting the captured nucleic acid from the mineral matrix, thereby isolating the nucleic acid.   
     
     
         15 . The method of  claim 13 , wherein the prepared sample comprises a final concentration (w/v) of the phase separation reagent of Formula I of about 0.05% to about 5.0%. 
     
     
         16 . The method of  claim 15 , wherein the prepared sample comprises a final concentration (w/v) of the phase separation reagent of Formula I of about 0.2% to about 0.3%. 
     
     
         17 . The method of  claim 13 , wherein the phase separation solution further comprises a salt solution selected from the group consisting of lithium salt, sodium salt, potassium salt, magnesium salt, or calcium salt, and combinations thereof. 
     
     
         18 . The method of  claim 13 , wherein the phase separation solution comprises domiphen bromide (DB). 
     
     
         19 . The method of  claim 13 , wherein the prepared sample further comprises a final concentration (M) of about 0.05 M to about 1.05 M lithium chloride or sodium chloride. 
     
     
         20 . The method of  claim 19 , wherein the final lithium chloride or sodium chloride concentration (M) is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 07, and 1.0 M. 
     
     
         21 . The method of  claim 19 , wherein the prepared sample comprises a final concentration (M) of about 0.1 M to about 0.65 M lithium chloride or sodium chloride. 
     
     
         22 . The method of  claim 21 , wherein the final lithium chloride or sodium chloride concentration (M) is about 0.1, 0.2, 0.3, 0.4, 0.5, or 0.6 M. 
     
     
         23 . The method of  claim 13 , further defined as a method for selectively capturing a phase separated nucleic acid. 
     
     
         24 . The method of  claim 23 , wherein the phase separation solution comprises domiphen bromide. 
     
     
         25 . The method of  claim 24 , wherein the prepared sample comprises a final concentration (M) of about 0.05 M to about 1.05 M lithium chloride or sodium chloride or about 0.1 M to about 0.65 M lithium chloride or sodium chloride. 
     
     
         26 . The method of  claim 21 , wherein the final lithium chloride or sodium chloride concentration (M) is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6. 
     
     
         27 . The method of  claim 26 , wherein the prepared sample comprises a final lithium chloride or sodium chloride concentration of about 0.1 M to about 0.7 M. 
     
     
         28 . The method of  claim 13 , further defined as a method for selectively capturing a phase separated genomic DNA. 
     
     
         29 . The method of  claim 28 , wherein the phase separation solution comprises domiphen bromide. 
     
     
         30 . The method of  claim 29 , wherein the prepared sample comprises a final concentration (M) of about 0.05 M to about 1.05 M LiCl or NaCl or about 0.6 M to about 0.7 M LiCl or NaCl. 
     
     
         31 . The method of  claim 13 , wherein the phase separation solution further comprises a buffer. 
     
     
         32 . The method of  claim 31 , wherein the buffer comprises a sodium acetate, Tris or Bis-Tris buffer. 
     
     
         33 . The method of  claim 32 , wherein the buffer comprises a Bis-Tris or a Tris buffer and the prepared sample comprises a final concentration (M) of about 0.1M to about 0.7M lithium chloride or sodium chloride. 
     
     
         34 . The method of  claim 31 , wherein the phase separation reagent comprises a pH between about 5.0 and about 8.5 or a pH of about 7.0-7.5. 
     
     
         35 . The method of  claim 34  wherein the phase separation reagent comprises a pH of about 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, or 8.0. 
     
     
         36 . The method of  claim 13 , wherein the mineral matrix is silica-based. 
     
     
         37 . The method of  claim 36 , wherein the mineral matrix is borosilicate glass fiber. 
     
     
         38 . A method of isolating plasmid DNA by alkaline lysis, comprising:
 (a) resuspending cells comprising plasmid DNA in a first aqueous solution;   (b) lysing a sample with a second solution;   (c) neutralizing the sample;   (d) capturing phase separated plasmid DNA to a mineral matrix with a phase separation solution comprising a cationic surfactant of Formula I   
       
         
           
           
               
               
           
         
         wherein:
 X 1  is nitrogen, phosphorus, N-heteroaryl (C≦18)  or substituted N-heteroaryl (C≦18) , provided that when X 1  is N-heteroaryl (C≦18)  or substituted N-heteroaryl (C≦18) , then R 2 , R 3 , and R 4  are absent and when R 2 , R 3 , and R 4  are absent, then X 1  is N-heteroaryl (C≦18)  or substituted N-heteroaryl (C≦18) ; 
 R 1  is alkyl (C≦30) , alkenyl (C≦30) , alkynyl (C≦30) , aryl (C≦30) , aralkyl (C≦30) , heteroaryl (C≦30) , heteroaralkyl (C≦30) , a substituted version of any of these groups or —Y 1 —Z—Y 2 ;
 Y 1  is alkandiyl (C≦6) , alkendiyl (C≦6) , alkyndiyl (C≦6) , arenediyl (C≦6) , alkoxydiyl (C≦6) , or a substituted version of any of these groups;
 Z is —O—, —NH—, —S—, —C(O)O—, —OC(O)—, —NHC(O)—;or —C(O)NH-; 
 
 
 
         Y 2  is alkyl (C≦30) , alkenyl (C≦30) , alkynyl (c≦30) , aryl (C≦30) , aralkyl (C≦30) , heteroaryl (C≦30) , heteroaralkyl (C≦30) , or a substituted version of any of these groups;
 R 2 , R 3 , and R 4  are each independently alkyl (C≦30) , alkenyl (C≦30) , alkynyl (C≦30) , aryl (C≦30) , aralkyl (C≦30) , heteroaryl (C≦30) , heteroaralkyl (C≦30) , a substituted version of any of these groups or —Y 3 —Z—Y 4 ;
 Y 3  is alkandiyl (C≦6) , alkendiyl (C≦6) , alkyndiyl (C≦6) , arenediyl (C≦6) , or a substituted version of any of these groups;
 Z is —O—, —NH—, —S—, —C(O)O—, —OC(O)—, —NHC(O)—; or —C(O)NH—; 
 
 Y 4  is alkyl (C≦30) , alkenyl (C≦30) , alkynyl (C≦30) , aryl (C≦30) , aralkyl (C≦30) , heteroaryl (C≦30) , heteroaralkyl (C≦30) , or a substituted version of any of these groups; 
 
 A is bromide, chloride, iodide, phosphate, sulfate, acetate, formate, propionate, oxalate, or succinate; 
 
         provided that when R 2 , R 3  and R 4  are alkyl (C≦30) , alkenyl (C≦30) , alkynyl (C≦30) , aryl (C≦30) , aralkyl (C≦30) , heteroaryl (C≦30) , heteroaralkyl (C≦30) , or a halo substituted version of any of these groups and X 1  is nitrogen or phosphorus, then R 1  is —Y 1 —Z—Y 2 ; 
         (e) treating the captured plasmid DNA with a salt solution, thereby enhancing the retention of captured nucleic acid; 
         (f) washing the captured plasmid DNA with an organic wash solution; and 
         (g) eluting the plasmid DNA, thereby isolating the plasmid DNA. 
       
     
     
         39 - 101 . (canceled)