Method and Materials for Quaternary Amine Catalyzed Bisulfite Conversion of Cytosine to Uracil

Abstract

The invention provides methods and materials for the conversion of cytosine to uracil. A nucleic acid, such a gDNA, is reacted with bisulfate, such as magnesium bisulfite, in the presence of a quaternary amine catalyst. Examples of suitable quaternary amine catalysts include but are not limited to quaternary ammonium compounds, quaternary alkyl ammonium salts, quaternary alkyl ammonium halides, quaternary methyl ammonium bromide, quaternary ammonium chloride, tetraethyl ammonium hydroxide, tetraethylammonium chloride, tetrabutyl ammonium chloride, tetrabutyl ammonium bromide. The invention also contemplates kits of premeasured ingredients for carrying out the methods of the invention either on an individual sample or on a plurality of samples.

1 . A method for converting cytosine to uracil comprising the steps of:
 providing a nucleic acid comprising at least one cytosine nucleobase; and   reacting the nucleic acid with a bisulfite ion, in the presence of a quaternary amine catalyst having the Formula:   
       
         
           
           
               
               
           
         
         or a derivative thereof, wherein: 
         R 1 , R 2 , R 3  and R 4  are each independently C 1 -C 4  alkyl; and
 Z is selected from halides and OH. 
 
       
     
     
         2 . The method of  claim 1 , wherein the nucleic acid is gDNA and further wherein a step of predenaturation of the gDNA prior to the step of reacting the gDNA with the bisulfite ion is not performed. 
     
     
         3 . The method of  claim 1 , wherein the quaternary amine catalyst comprises at least one of quaternary methyl ammonium bromide, tetraethylammonium hydroxide, tetraethylammonium chloride, tetrabutylammonium chloride and tetrabutylammonium bromide. 
     
     
         4 . The method of  claim 1 , wherein the catalyst comprises tetraethylammonium hydroxide. 
     
     
         5 . The method of  claim 4  wherein the tetraethylammonium hydroxide is provided as an approximately 20% solution. 
     
     
         6 . The method of  claim 1  wherein the bisulfite ion comprises magnesium bisulfite. 
     
     
         7 . The method of  claim 6  wherein the magnesium bisulfite is provided as an approximately 2M solution. 
     
     
         8 . The method of  claim 7  wherein the final concentration of the magnesium bisulfite in the reaction mixture is approximately 1.3M. 
     
     
         9 . The method of  claim 1  wherein the reaction is performed at about 50° C. for about 4 to about 15 hours. 
     
     
         10 . The method of  claim 1 , further comprising treating the product of the reaction of the nucleic acid and the bisulfite ion with NaOH. 
     
     
         11 . The method of  claim 11 , wherein the NaOH is provided as an approximately 0.1M solution. 
     
     
         12 . A kit for use in conversion of cytosine to uracil in a nucleic acid comprising: magnesium bisulfite; and a quaternary amine catalyst. 
     
     
         13 . The kit of  claim 12 , wherein the magnesium bisulfite is provided as an approximately 2M solution. 
     
     
         14 . The kit of  claim 12  wherein the quaternary amine catalyst comprises at least one of quaternary methyl ammonium bromide, tetraethyl ammonium hydroxide, tetraethyl ammonium chloride, tetrabutyl ammonium chloride and tetrabutyl ammonium bromide. 
     
     
         15 . The kit of  claim 14  wherein the quaternary amine catalyst comprises tetraethylene ammonium hydroxide. 
     
     
         16 . The kit of  claim 15  wherein the tetraethyl ammonium hydroxide is provided as an approximately 20% solution. 
     
     
         17 . The kit of  claim 12  further comprising NaOH, wherein the NaOH is provided as an approximately 0.1M solution. 
     
     
         18 . The kit of  claim 12  further comprising a nucleotide polymerase and one or more primers for sequencing or amplification. 
     
     
         19 . The kit of  claim 12  further comprising one or more reaction vessels. 
     
     
         20 . The kit of  claim 12  further comprising prepackaged materials sufficient to convert cytosine to uracil in one or more nucleic acid samples containing at least one cytosine nucleobase.