Methods and kits for detection of 5-hydroxymethylcytosine

Abstract

The present invention relates to methods and kits for the detection of 5-hydroxymethylcytosine (5hmC). In some embodiments, the present invention relates to methods and kits for detection of 5hmC in nucleic acid (e.g., DNA, RNA). In some embodiments, the present invention relates to detection of 5hmC in genomic DNA, e.g., mammalian genomic DNA.

1 . A method for detecting 5-hydroxymethylcytosine in a sample comprising nucleic acid comprising:
 a) glucosylating 5-hydroxymethylcytosine bases in said sample to provide nucleic acid comprising glucosylated 5-hydroxymethylcytosine bases; and   b) contacting said sample with a binding agent specific for glycosylated 5-hydroxymethylcytosine to form a binding agent-modified 5-hydroxymethylcytosine complex, wherein said binding agent is a  C. fasciculata  J binding protein immobilized on a substrate.   
     
     
         2 - 7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein said biological sample is obtained from a subject selected from the group consisting of a mammal, plant, fish, bird, fungus and bacteria. 
     
     
         9 . The method of  claim 8 , wherein said mammal is a human. 
     
     
         10 . The method of  claim 9 , wherein said glycosylation is accomplished by a glucosyltransferase. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 10 , wherein said glucosyltransferase is a beta-glucosyltransferase. 
     
     
         13 . The method of  claim 12 , wherein said beta-glucosyltransferase is a T4 bacteriophage beta-glycosyltransferase. 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein said solid substrate is selected from the group consisting of a particulate material and a nonporous material. 
     
     
         16 . The method of  claim 15 , wherein said particulate material is selected from the group consisting of a bead, a colloid, a gel, and a magnetic particle. 
     
     
         17 . The method of  claim 15 , wherein said nonporous material is selected from the group consisting of a well, a plate, and a membrane. 
     
     
         18 . The method of  claim 1 , further comprising the step of analyzing said binding agent-glucosylated 5 hydroxymethylcytosine complex. 
     
     
         19 . The method of  claim 18 , wherein said analyzing comprises a technique selected from the group consisting of PCR, real-time PCR, mass spectrometry, hybridization, gene arrays, and DNA sequencing. 
     
     
         20 . The method of  claim 19 , further comprising comparing the presence of 5-hydroxymethylcytosine in said nucleic acid in said sample to a reference standard, wherein an increased or decreased level of 5-hydroxymethylcytosine in said nucleic acid is indicative of the presence of a disease or of the probable course of a disease. 
     
     
         21 . The method of  claim 20 , further comprising the step of providing a diagnoses or prognoses based on an increased or decreased level of 5-hydroxymethylcytosine in said nucleic acid as compared to a reference standard. 
     
     
         22 . The method of  claim 20 , wherein said disease is cancer.