Rapid purification of high quality nucleic acids from biological samples

Abstract

This invention relates to a method for the purification of nucleic acids, preferably DNA, from biological samples, comprising the steps (a) optional lysis of said sample, (b) optional heat incubation of said sample, (c) enzymatic digestion of non-nucleic acid components in the product of step (a) or (b), (d) heat inactivation of one or more enzyme(s) used in step (c), (e) transfer of the product of step (d) onto a resin capable of retaining non-nucleic acid components, while the nucleic acids pass through the resin, thereby purifying the nucleic acids.

1 . A method for isolating nucleic acids from a sample, comprising the following steps:
 (a) lysis of said sample in the presence of at least 0.1 mM EDTA and a detergent;   (b) enzymatic digestion of non-nucleic acid components in the product of step (a);   (c) heat inactivation of one or more enzyme(s) used in step (b);   (d) transfer of the product of step (c) onto a resin capable of retaining non-nucleic acid components, while the nucleic acids pass through the resin;   thereby purifying the nucleic acids.   
     
     
         2 . The method of  claim 1 , wherein the enzymatic digestion of step (b) is carried out for 1 to 240 min, optionally for 5 to 120 min. 
     
     
         3 . The method of  claim 1 , wherein step (a) is conducted in the presence of SDS, optionally to a final concentration of at least 20 mM SDS, at least 60 mM SDS, at least 100 mM SDS, at least 150 mM SDS or 200 mM SDS. 
     
     
         4 . The method of  claim 1 , wherein the enzymatic digestion of step (b) is carried out for at least 1 min, at least 5 min, at least 10 min, at least 15 min or at least 20 min. 
     
     
         5 . The method of  claim 1 , wherein one or more enzyme(s) is/are used in step (b) for the enzymatic digestion, wherein the one or more enzyme(s) is/are lytic enzyme(s), selected from the group consisting of proteases, lipases, cellulases, hydrolases, chitinases, amylases and glucanases, wherein hydrolases are not nucleases. 
     
     
         6 . The method of  claim 1 , wherein the enzymatic digestion of step (b) is carried out at 60° C. for 5 min to 120 min, optionally for 30 mins. 
     
     
         7 . The method of  claim 1 , wherein one or more protease(s) is/are used in step (b) and wherein the one or more protease(s) is/are selected from the group consisting of protease from  Bacillus licheniformis , protease from  Bacillus  spec., protease from  Staphylococcus aureus , protease from  Bacillus amyloliquefaciens , protease from  Coprinus  spec. and protease from  Aspergillus oryzae.    
     
     
         8 . The method of  claim 1 , wherein ammonium salt(s) is/are added in step (a), optionally to a final concentration of at least 5 mM. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein SrCl 2  and/or BaCl 2  and/or RbCl and/or CsCl and/or CaCl 2  is/are added to the product of step (c) before transfer onto the resin in step (d). 
     
     
         11 . The method of  claim 1 , wherein a chelating agent is present at step (a), optionally wherein the chelating agent is EDTA, optionally wherein the chelating agent is present at a concentration of at least 0.1 mM. 
     
     
         12 . The method of  claim 1 , wherein said sample is a feces sample, blood sample, urine sample, tissue sample and/or body fluid sample. 
     
     
         13 . The method of  claim 1 , wherein said nucleic acid is DNA and/or RNA, optionally DNA. 
     
     
         14 . The method of  claim 1 , wherein a centrifugation step is executed in step (d) after transfer of the product of step (c) onto the resin at 400 g to 3000 g for 0.5 min to 5 min, optionally for about 1 min. 
     
     
         15 . The method of  claim 1 , wherein said resin is a size-exclusion resin with exclusion limits in the range of 20 to 2000 bp of single and/or double stranded nucleotide strands. 
     
     
         16 . The method of  claim 1 , wherein said resin is incorporated into a spin column. 
     
     
         17 . The method of  claim 1 , wherein the resin is centrifuged for at least 1 min at a force of at least 300 g before the lysate of step (c) is added onto the resin or the spin column in step (d). 
     
     
         18 . The method of  claim 1 , wherein the lysate of step (b) or step (c) is cleared from precipitates by centrifugation of the lysate before it is applied onto the resin in step (d), preferably by centrifugation at about 10.000 g for 1 to 5 min. 
     
     
         19 - 20 . (canceled) 
     
     
         21 . The method of  claim 1 , wherein the detergent comprises SDS, derivatives of SDS, Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octyl glucoside, octyl thioglucoside, CHAPS or CHAPSO. 
     
     
         22 . The method of  claim 1 , wherein the detergent comprises SDS or its derivatives. 
     
     
         23 . The method of  claim 22 , wherein the detergent comprises LiDS. 
     
     
         24 . The method of  claim 10 , wherein the SrCl 2  and/or BaCl 2  and/or RbCl and/or CsCl and/or CaCl 2  is/are added to the product of step (c) to a final concentration of at least 400 mM. 
     
     
         25 . A kit comprising:
 a lysis buffer, comprising:
 a detergent comprising SDS or its derivatives; 
 a quantity of EDTA having a concentration of at least 0.1 mM; 
   a solution for the precipitation of non-nucleic components comprising SrCl 2  and/or BaCl 2  and/or RbCl and/or CsCl and/or CaCl 2 ; and   a size-exclusion resin.   
     
     
         26 . The kit according to  claim 25 , wherein the detergent comprises LiDS.