Methods and kits for detection of methylation status

Abstract

The present invention relates to methods and kits for the detection of 5-hydroxymethylcytosine (5hmC) and/or 5-methylcytosine (5meC). In some embodiments, the present invention relates to methods and kits for detection of 5hmC and/or 5meC 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 .- 29 . (canceled) 
     
     
         30 . A method comprising:
 (a) providing a polynucleotide comprising a first sequence and a second sequence complementary to said first sequence, wherein said first sequence comprises a nucleotide that is cytosine or modified cytosine;   (b) modifying said nucleotide in said first sequence, thereby generating a transformed first sequence;   (c) sequencing (i) said first transformed sequence or derivative thereof and (ii) said second sequence or derivative thereof to obtain: (i) a first determined sequence corresponding to said first transformed sequence or derivative thereof and (ii) a second determined sequence corresponding to said second sequence or derivative thereof; and   (d) comparing said first determined sequence and said second determined sequence to identify said nucleotide as cytosine or modified cytosine.   
     
     
         31 . The method of  claim 30 , further comprising, prior to (a), contacting a precursor polynucleotide with a DNA-glucosyltransferase to generate said polynucleotide. 
     
     
         32 . The method of  claim 30 , further comprising, prior to (a), contacting a precursor polynucleotide with a methyltransferase to generate said polynucleotide. 
     
     
         33 . The method of  claim 30 , wherein (b) comprises contacting said first sequence with a methyltransferase enzyme. 
     
     
         34 . The method of  claim 30 , wherein (b) comprises contacting said first sequence with a TET enzyme. 
     
     
         35 . The method of  claim 30 , wherein (b) comprises contacting said first sequence with a DNA-glucosyltransferase. 
     
     
         36 . The method of  claim 30 , further comprising, prior to (a), amplifying a precursor polynucleotide to generate said polynucleotide. 
     
     
         37 . The method of  claim 36 , wherein said amplifying is performed with a tagged primer. 
     
     
         38 . The method of  claim 30 , wherein, in (a), said nucleotide is said cytosine, and wherein said first sequence comprises an additional nucleotide that is a modified cytosine. 
     
     
         39 . The method of  claim 38 , wherein (b) comprises modifying both said nucleotide and said additional nucleotide to generate said transformed first sequence. 
     
     
         40 . The method of  claim 39 , wherein (d) comprises comparing said first determined sequence and said second determined sequence to distinguish said nucleotide as cytosine and said additional nucleotide as modified cytosine. 
     
     
         41 . The method of  claim 30 , wherein the modified cytosine is a methylated cytosine or a hydroxymethylated cytosine. 
     
     
         42 . The method of  claim 41 , wherein said modified cytosine is said hydroxymethlated cytosine, and wherein said hydroxymethylated cytosine comprises 5-hydroxymethyl cytosine or β-glu-5-hydroxymethyl cytosine. 
     
     
         43 . The method of  claim 30 , wherein said first determined sequence comprises a thymidine at a first base corresponding to said nucleotide, wherein said second determined sequence comprises a cytosine at a second base corresponding to said first base, and wherein (d) comprises comparing said first determined sequence and said second determined sequence to identify said nucleotide as 5-hydroxymethylcytosine. 
     
     
         44 . The method of  claim 30 , wherein said first determined sequence comprises a cytosine at a first base corresponding to said nucleotide, wherein said second determined sequence comprises a cytosine at a second base corresponding to said first base, and wherein (d) comprises comparing said first determined sequence and second determined sequence to identify said nucleotide as a 5-methylcytosine. 
     
     
         45 . The method of  claim 30 , wherein said first determined sequence comprises a thymidine at a first base corresponding to said nucleotide, wherein said second determined sequence comprises a thymine at a second base corresponding to said first base, and wherein (d) comprises comparing said first determined sequence, and second determined sequence to identify said nucleotide as cytosine. 
     
     
         46 . The method of  claim 30 , wherein (b) comprises modifying an additional nucleotide in said second sequence thereby generating a transformed second sequence, and wherein (c) comprises sequencing said transformed second sequence or derivative thereof to obtain said second determined sequence. 
     
     
         47 . The method of  claim 30 , further comprising: (i) comparing an identity of the nucleotide identified in (d) to a reference, and (ii) based on said comparing of (i) identifying a presence of a disease or condition or the probable course of a disease or condition. 
     
     
         48 . The method of  claim 47 , wherein said disease or condition comprises cancer. 
     
     
         49 . The method of  claim 30 , wherein said cytosine or modified cytosine is identified at base specific resolution. 
     
     
         50 . The method of  claim 30 , wherein, in (c), said sequencing is performed by a next generation sequencing method. 
     
     
         51 . The method of  claim 30 , wherein (b) comprises contacting said polynucleotide with an oxidizing agent. 
     
     
         52 . The method of  claim 30 , further comprising contacting said polynucleotide with a tagged oligonucleotide.