COMPOSITION AND METHODS RELATED TO MODIFICATION OF 5 HYDROXYMETHYLCYTOSINE (5-hmC)

Abstract

The present invention relates generally to the field of molecular biology. More particularly, it concerns methods and compositions for detecting, evaluating, and/or mapping 5-hydroxymethyl-modified cytosine bases within a nucleic acid molecule.

1 .- 171 . (canceled) 
     
     
         172 . A kit comprising purified β-glucosyltransferase and a synthetically modified uridine diphosphoglucose (UDP-Glu) molecule, a polymerase, and one or more nucleic acid buffers, wherein the synthetically modified UDP-Glu comprises a synthetic linker, a detectable moiety, an isolation tag, a blocking moiety, or a functional moiety. 
     
     
         173 . A method for detecting 5-hydroxymethylcytosine in an isolated nucleic acid sample comprising:
 a) incubating the nucleic acid sample with β-glucosyltransferase and a modified uridine diphosphoglucose (UDP-Glu) molecule, wherein a modified Glu is enzymatically attached to 5-hydroxymethylcytosines in nucleic acid molecules in the nucleic acid sample;   b) detecting the 5-hydroxymethylcytosines in the nucleic acid sample based on the presence of the modified Glu to determine the 5-hydroxymethylcytosine status of the nucleic acid sample as compared to the 5-hydroxymethylcytosine status of a control nucleic acid sample.   
     
     
         174 . The method of  claim 173 , wherein the isolated nucleic acid sample comprises genomic DNA. 
     
     
         175 . The method of  claim 173 , wherein the isolated nucleic acid sample is on an array. 
     
     
         176 . The method of  claim 175 , wherein the array is a microarray. 
     
     
         177 . The method of  claim 173 , wherein the modified UDP-Glu comprises a linker and/or detectable moiety. 
     
     
         178 . The method of  claim 177 , wherein the linker is an azide linker. 
     
     
         179 . The method of  claim 177 , wherein the linker is a thiol linker. 
     
     
         180 . The method of  claim 177 , wherein the detectable moiety is fluorescent, radioactive, enzymatic, electrochemical, or colorimetric. 
     
     
         181 . The method of  claim 180 , wherein the detectable moiety is a fluorophore. 
     
     
         182 . The method of  claim 173 , wherein the modified UDP-Glu comprises an isolation tag. 
     
     
         183 . The method of  claim 182 , wherein the isolation tag is biotin or a histidine tag. 
     
     
         184 . The method of  claim 173 , wherein detecting the 5-hydroxymethylcytosines in the nucleic acid sample comprises sequencing the glycosylated nucleic acid. 
     
     
         185 . The method of  claim 173 , wherein detecting the 5-hydroxymethylcytosines in the nucleic acid sample comprises subjecting the glycosylated nucleic acid to a primer extension assay. 
     
     
         186 . The method of  claim 173 , wherein detecting the 5-hydroxymethylcytosines in the nucleic acid sample comprises subjecting the glycosylated nucleic acid to a hybridization assay. 
     
     
         187 . The method of  claim 173 , wherein the nucleic acid sample is from a human patient. 
     
     
         188 . A method for detecting 5-hydroxymethylcytosine in a genomic DNA sample from a patient comprising:
 a) incubating the sample with β-glucosyltransferase and a modified uridine diphosphoglucose (UDP-Glu) molecule to attach the modified Glu to 5-hydroxymethylcytosines in the genomic DNA;   b) detecting 5-hydroxymethylcytosines in the genomic DNA from the patient based on the presence of the modified Glu to determine the 5-hydroxymethylcytosine status of the nucleic acid sample as compared to the 5-hydroxymethylcytosine status of a control nucleic acid sample.   
     
     
         189 . The method of  claim 188 , wherein detecting the 5-hydroxymethylcytosines in the genomic DNA comprises sequencing the glycosylated genomic DNA. 
     
     
         190 . The method of  claim 188 , wherein detecting the 5-hydroxymethylcytosines in the genomic DNA comprises subjecting the glycosylated DNA to a primer extension assay. 
     
     
         191 . The method of  claim 188 , wherein detecting the 5-hydroxymethylcytosines in the genomic DNA comprises subjecting the glycosylated DNA to a hybridization assay.