Bisulfite conversion of DNA

Abstract

The present invention provides an improved method for the bisulfite conversion of DNA, and facilitates the analysis of cytosine methylation of genomic DNA. Novel combinations of denaturing solvents, new reaction conditions and new purification methods provide surprisingly efficacious methods for bisulfite conversion of DNA relative to prior art methods. The converted DNA may subsequently be analyzed by many different methods.

1 . A method for bisulfite conversion of DNA, comprising reacting genomic DNA with a bisulfite reagent, wherein said reaction is carried out in the presence of a compound selected from the group consisting of dioxane, one of its derivatives, and a similar aliphatic cyclic ether.  
     
     
         2 . The method according to  claim 1 , wherein the concentration of said compounds amounts to about 10-35%.  
     
     
         3 . The method according to  claim 1 , wherein the concentration of said compounds amounts to about 20-30%.  
     
     
         4 . A method for bisulfite conversion of DNA, comprising reacting genomic DNA with a bisulfite reagent, wherein said reaction is carried out in the presence of a compound with the following formula:  
       
         
           
           
               
               
           
         
         n=1-35000  
         m=1-3  
         R 1 =H, Me, Et, Pr, Bu  
         R 2 =H, Me, Et, Pr, Bu  
       
     
     
         5 . The method according to  claim 4 , wherein said compound comprises an n-alkylene glycol.  
     
     
         6 . The method according to  claim 5 , wherein said compound comprises a dialkyl ether.  
     
     
         7 . The method according to  claim 6 , where said compound comprises diethylene glycol dimethyl ether (DME).  
     
     
         8 . The method according to  claim 4 , wherein said compound is present in a concentration of about 1-35%.  
     
     
         9 . The method according to  claim 4 , wherein said compound is present in a concentration of about 5-25%.  
     
     
         10 . A method for bisulfite conversion of DNA, comprising reacting isolated genomic DNA with a bisulfite reagent, wherein the reaction is conducted at a temperature in the range of 0-80° C., and wherein the reaction temperature is briefly increased to a range of about 85-100° C. during the course of the conversion.  
     
     
         11 . The method according to  claim 10 , wherein the number of temperature increases of brief duration amounts to 2 to 5.  
     
     
         12 . The method according to  claim 10 , wherein during the temperature increases of brief duration, the reaction temperature increases to about 85 to 100° C.  
     
     
         13 . The method according to  claim 12 , wherein the temperature increases of brief duration increase the reaction temperature to about 90 to 98° C.  
     
     
         14 . The method according to  claim 10 , wherein the converted DNA is purified via magnetic particles.  
     
     
         15 . A method for the bisulfite conversion of DNA, comprising reacting DNA with a bisulfite reagent, and purifying the converted DNA by means of ultrafiltration.  
     
     
         16 . The method according to any one of claims  1 ,  4 ,  10  or  15 , wherein the converted DNA is analyzed, at least in part, by using at least one method selected from the group consisting of: MSP, HeavyMethyl, MsSNuPE and MethylLight.  
     
     
         17 . The method according to any one of claims claims  1 ,  4 ,  10  or  15 , wherein DNA of tissue samples or bodily fluids is investigated.  
     
     
         18 . A method for at least one of diagnosis and prognosis of an adverse event for patients or individuals, comprising use of a method according to any one of claims  1 ,  4 ,  10  or  15 , wherein the adverse event is at least one event selected from the category group consisting of: undesired drug interactions; cancer diseases; CNS malfunctions, damage or disease; symptoms of aggression or behavioral disturbances; clinical, psychological and social consequences of brain damage; psychotic disturbances and personality disorders; dementia and/or associated syndromes; cardiovascular disease, malfunction and damage; malfunction, damage or disease of the gastrointestinal tract; malfunction, damage or disease of the respiratory system; lesion, inflammation, infection, immunity and/or convalescence; malfunction, damage or disease of the body as an abnormality in the development process; malfunction, damage or disease of the skin, of the muscles, of the connective tissue or of the bones; endocrine and metabolic malfunction, damage or disease; headaches or sexual malfunction.  
     
     
         19 . A method for distinguishing cell types or tissues, or for investigating cell differentiation, comprising use of a method according to any one of claims  1 ,  4 ,  10  or  15 .  
     
     
         20 . A kit, comprising: a reagent containing bisulfite; denaturing reagents or solvents, a radical scavenger, and primers for the production of amplificates.  
     
     
         21 . The kit of  claim 20 , further comprising at least one of an ultrafiltration tube, and instructions for conducting an assay according to any one of the preceding claims.