Compositions and methods for obtaining nucleic acids from sputum

Abstract

The present invention relates to compositions and methods for preserving and extracting nucleic acids from saliva. The compositions include a chelating agent, a denaturing agent, buffers to maintain the pH of the composition within ranges desirable for DNA and/or RNA. The compositions may also include a reducing agent and/or antimicrobial agent. The invention extends to methods of using the compositions of the invention to preserve and isolate nucleic acids from saliva as well as to containers for the compositions of the invention.

1 . A method for preserving nucleic acid contained in a biological sample comprising the steps of:
 a) obtaining the biological sample from a subject; and   b) contacting said sample with a composition comprising
 (i) at least one chelating agent selected from the group consisting of chelating agents stronger than EDTA; and 
 (ii) a denaturing agent, wherein the pH of said composition is greater than 5.0, 
 to form a liquid mixture; and 
   c) storing said mixture for more than 1 day at room temperature,   wherein said composition stabilizes said nucleic acid for more than 1 day at room temperature.   
   
   
       2 . The method of  claim 1 , wherein the biological sample is tissue or a bodily fluid. 
   
   
       3 . The method of  claim 2 , wherein the bodily fluid is sputum. 
   
   
       4 . The method of  claim 3 , wherein the sputum is saliva. 
   
   
       5 . The method of  claim 1 , wherein said chelating agent is cyclohexane diaminotetraacetate (CDTA), diethylenetriamine pentaacetic acid (DTPA), tetraazacycylododecanetetraacetic acid (DOTA), tetraazacyclotetradecanetetraacetic acid (TETA), desferioximine or a chelator analog thereof. 
   
   
       6 . The method of  claim 1 , wherein said denaturing agent is urea, dodecyl sulfate, guanidinium chloride, guanidinium thiocyanate, perchlorate, an alcohol or a mixture thereof. 
   
   
       7 . The method of  claim 6 , wherein the alcohol is ethanol. 
   
   
       8 . The method of  claim 1 , wherein the chelating agent is CDTA and the denaturing agent is SDS. 
   
   
       9 . The method of  claim 1 , wherein the pH is between 7.0 and 10.0 inclusive. 
   
   
       10 . The method of  claim 1 , wherein the biological sample is from a mammal. 
   
   
       11 . The method of  claim 1 , wherein the nucleic acid is from a virus, a bacterium or from said subject. 
   
   
       12 . The method of  claim 1 , further comprising the step of recovering said nucleic acid by contacting said nucleic acid-containing solution with a protease. 
   
   
       13 . The method of  claim 1 , wherein the nucleic acid is DNA. 
   
   
       14 . A method for recovering nucleic acid from a biological sample comprising the steps of:
 a) obtaining the biological sample from a subject; and   b) contacting said sample with a composition comprising
 (i) at least one chelating agent selected from the group consisting of chelating agents stronger than EDTA; and 
 (ii) a denaturing agent, wherein the pH of said composition is greater than 5.0, 
 to form a liquid mixture; and 
   c) storing said mixture for more than 1 day at room temperature,   wherein said composition stabilizes said nucleic acid for more than 1 day at room temperature;   d) contacting said mixture with a protease; and   e) recovering said nucleic acid from said mixture.   
   
   
       15 . The method of  claim 14 , wherein the biological sample is tissue or a bodily fluid. 
   
   
       16 . The method of  claim 15 , wherein the bodily fluid is sputum. 
   
   
       17 . The method of  claim 16 , wherein the sputum is saliva. 
   
   
       18 . The method of  claim 14 , wherein said chelating agent is cyclohexane diaminotetraacetate (CDTA), diethylenetriamine pentaacetic acid (DTPA), tetraazacycylododecanetetraacetic acid (DOTA), tetraazacyclotetradecanetetraacetic acid (TETA), desferioximine or a chelator analog thereof. 
   
   
       19 . The method of  claim 14 , wherein said denaturing agent is urea, dodecyl sulfate, guanidinium chloride, guanidinium thiocyanate, perchlorate, an alcohol or a mixture thereof. 
   
   
       20 . The method of  claim 19 , wherein the alcohol is ethanol. 
   
   
       21 . The method of  claim 14 , wherein the chelating agent is CDTA and the denaturing agent is SDS. 
   
   
       22 . The method of  claim 14 , wherein the pH is between 7.0 and 10.0 inclusive. 
   
   
       23 . The method of  claim 14 , wherein the biological sample is from a mammal. 
   
   
       24 . The method of  claim 14 , wherein the nucleic acid is from a virus, a bacterium or from said subject. 
   
   
       25 . The method of  claim 14 , wherein the nucleic acid is DNA. 
   
   
       26 . The method according to  claim 14 , wherein the protease is proteinase K. 
   
   
       27 . A method for preserving nucleic acid contained in a biological sample comprising the steps of:
 a) obtaining the biological sample from a subject; and   b) contacting said sample with a composition comprising
 (i) at least one chelating agent selected from the group consisting of chelating agents stronger than EDTA; and 
 (ii) a denaturing agent, wherein the pH of said composition is greater than 5.0, 
 to form a liquid mixture; 
   c) contacting said mixture with a protease;   d) storing said mixture for more than 1 day at room temperature,   
     wherein said composition stabilizes said nucleic acid for more than 1 day at room temperature. 
   
   
       28 . The method of  claim 27 , wherein the biological sample is tissue or a bodily fluid. 
   
   
       29 . The method of  claim 28 , wherein the bodily fluid is sputum. 
   
   
       30 . The method of  claim 29 , wherein the sputum is saliva. 
   
   
       31 . The method of  claim 27 , wherein said chelating agent is cyclohexane diaminotetraacetate (CDTA), diethylenetriamine pentaacetic acid (DTPA), tetraazacycylododecanetetraacetic acid (DOTA), tetraazacyclotetradecanetetraacetic acid (TETA), desferioximine or a chelator analog thereof. 
   
   
       32 . The method of  claim 27 , wherein said denaturing agent is urea, dodecyl sulfate, guanidinium chloride, guanidinium thiocyanate, perchlorate, an alcohol or a mixture thereof. 
   
   
       33 . The method of  claim 32 , wherein the alcohol is ethanol. 
   
   
       34 . The method of  claim 27 , wherein the chelating agent is CDTA and the denaturing agent is SDS. 
   
   
       35 . The method of  claim 27 , wherein the pH is between 7.0 and 10.0 inclusive. 
   
   
       36 . The method of  claim 27 , wherein the biological sample is from a mammal. 
   
   
       37 . The method of  claim 27 , wherein the nucleic acid is from a virus, a bacterium or from said subject. 
   
   
       38 . The method of  claim 27 , wherein the nucleic acid is DNA.