Treatment of nucleic acid

Abstract

The invention provides methods for treating nucleic acid, particularly nucleic acid that is methylated. In one embodiment, the method can include the steps of (a) providing a denaturing environment to a nucleic acid sample; (b) reacting the nucleic acid sample with a bisulphite reagent and incubating the reaction so as to form a treated nucleic acid sample where methylated nucleotides in the nucleic acid sample remain unchanged while unmethylated nucleotides are converted to another form; (c) diluting the treated nucleic acid sample so as to reduce salt concentration to a level which will not substantially interfere with a nucleic acid precipitating step; (d) precipitating the diluted treated nucleic acid to substantially remove any unwanted reagents or diluents from treated nucleic acid; and (e) carrying out de-sulphonation of the precipitated treated nucleic acid so as to remove sulphonate groups present on the treated nucleic acid so as to obtain a nucleic acid sample substantially free of sulphonate groups.

1 - 27 . (canceled)  
     
     
         28 . A method for treating nucleic acid comprising: 
 (a) providing a denaturing environment to a nucleic acid sample;    (b) reacting the nucleic acid sample with a bisulphite reagent and incubating the reaction so as to form a treated nucleic acid sample where methylated nucleotides in the nucleic acid sample remain unchanged while unmethylated nucleotides are converted to another form;    (c) diluting the treated nucleic acid sample so as to reduce salt concentration to a level which will not substantially interfere with a nucleic acid precipitating step;    (d) precipitating the diluted treated nucleic acid to substantially remove any unwanted reagents or diluents from the treated nucleic acid sample; and    (e) carrying out de-sulphonation of the precipitated treated nucleic acid so as to remove sulphonate groups present on the treated nucleic acid so as to obtain a nucleic acid sample substantially free of sulphonate groups.    
     
     
         29 . The method according to  claim 28  wherein more than about 50% of the starting nucleic acid in the sample is retained.  
     
     
         30 . The method according to  claim 29  wherein more than about 75% of the starting nucleic acid in the sample is retained.  
     
     
         31 . The method according to  claim 30  wherein more than about 95% of the starting nucleic acid in the sample is retained.  
     
     
         32 . The method according to  claim 28  further comprising: 
 (f) processing or analysing the treated nucleic acid sample.    
     
     
         33 . The method according to  claim 28  wherein the sample comprises DNA, the sample comprises RNA, or the sample comprises combination of both DNA and RNA.  
     
     
         34 . The method according to  claim 28  wherein the sample is prepared from tissue, organ, cell, microorganism, biological sample, or environmental sample.  
     
     
         35 . The method according to  claim 28  carried out in a reaction vessel. The method according to claim I wherein the bisulphite reagent is sodium metabisulphite.  
     
     
         36 . The method according to  claim 35  wherein the reaction vessel is selected from the group consisting of tube, plate, capillary tube, well, centrifuge tube, microfuge tube, slide, coverslip, and surface.  
     
     
         37 . The method according to  claim 28  wherein the denaturing environment is provided to the sample by adding an alkali.  
     
     
         38 . The method according to  claim 28  wherein the denaturing environment is provided to the sample by adding an alkali.  
     
     
         39 . The method according to  claim 38  wherein the alkali is NaOH, KOH, or any compound or agent providing hydroxyl groups.  
     
     
         40 . The method according to  claim 28  wherein the denaturing environment is provided to the sample by heating.  
     
     
         41 . The method according to  claim 40  wherein the heating is up to about 95° C.  
     
     
         42 . The method according to  claim 41  wherein the heating is from 50° C. to 70° C.  
     
     
         43 . The method according to  claim 28  wherein the providing step (a) is carried out in the presence of an additive capable of enhancing the bisulphite reaction.  
     
     
         44 . The method according to  claim 43  wherein the additive is selected from the group consisting of quinol, urea, methoxyamine, and mixtures thereof.  
     
     
         45 . The method according to  claim 28  wherein the reacting step (b) results in methylated cytosines in the nucleic acid sample remaining unchanged while unmethylated cytosines are converted to uracils.  
     
     
         46 . The method according to  claim 28  wherein the dilution step (c) is carried out using water to reduce salt concentration to below about 0.5 M.  
     
     
         47 . The method according to  claim 28  wherein the precipitation is carried out using an alcohol precipitating agent.  
     
     
         48 . The method according to  claim 47  wherein the alcohol precipitating agent is selected from the group consisting of isopropanol, ethanol, butanol, methanol, and mixtures thereof.  
     
     
         49 . The method according to  claim 48  wherein the alcohol is isopropanol.  
     
     
         50 . The method according to  claim 28  wherein the de-sulphonation step (d) is carried out by adjusting the precipitated treated nucleic acid up to about pH 12.5 with a buffer or alkaline reagent.  
     
     
         51 . The method according to  claim 50  wherein the pH is adjusted to about 10.5.  
     
     
         52 . A kit for carrying out the method according to  claim 28  comprising vessels containing reagents and instructions to use the reagents.  
     
     
         53 . The kit according to  claim 52  wherein the reagents comprise a diluent, a bisulphite reagent and an alkali.  
     
     
         54 . The kit according to  claim 53  wherein the bisulphite reagent is sodium metabisulphite.