Detection and Quantification of Hydroxymethylated Nucleotides in a Polynucleotide Preparation

Abstract

Methods and compositions are described for detecting hydroxymethylated nucleotides (hmNs) in a polynucleotide preparation with a view to mapping the location of hmNs in a genome, quantifying the occurrence of hmNs at selected loci and correlating the occurrence of hmNs with gene expression and phenotypic traits. Embodiments describe the use of modifying enzymes together with site-specific endonucleases to detect the hmNs.

What is claimed is: 
     
         1 . A method of detecting a hydroxymethylated nucleotide (hmN) in a polynucleotide preparation; comprising:
 (a) obtaining a polynucleotide preparation; in which an hmN is modified;   (b) reacting polynucleotides in the polynucleotide preparation with a site-specific endonuclease which is capable of cleaving a polynucleotide at a specific recognition site containing at least a methylated nucleotide (mN) or hydroxymethylated nucleotide (hmN) but not a modified hmN (mhmN);   (c) detecting an uncleaved polynucleotides that would otherwise be cleaved but for the modified hmN; so as to determine the presence of the hmN in the polynucleotide preparation.   
     
     
         2 . A method according to  claim 1 , wherein (c) further comprises detecting a cleaved polynucleotide in the polynucleotide preparation. 
     
     
         3 . A method according to  claim 1 , wherein (a) further comprises ligating an adapter to the polynucleotide preparation for amplifying or sequencing an uncleaved polynucleotide. 
     
     
         4 . A method according to  claim 1 , wherein (c) further comprises mapping an identified hmNs to a genomic locus. 
     
     
         5 . A method according to  claim 1 , wherein the polynucleotide preparation is derived from a cell, tissue or organism and wherein (c) further comprises identifying an hmN at a predetermined locus in a genome for the polynucleotide in the preparation. 
     
     
         6 . A method according to  claim 1 , further comprising determining an amount of the hmN in the predetermined locus in the genome from a cell, a tissue or an organism. 
     
     
         7 . A method according to  claim 1 , further comprising: comparing the amount of hmN in polynucleotides in a first polynucleotide preparation and for polynucleotides in a second polynucleotide preparation. 
     
     
         8 . A method according to  claim 1 , further comprising: correlating with a phenotypic trait, a difference in the amount of the hmN at a predetermined locus in a first polynucleotide in a first polynucleotide preparation and in a second polynucleotide in a second polynucleotide preparation. 
     
     
         9 . A method according to  claim 1 , wherein (b) further comprises reacting the polynucleotide preparation with a PvuRts1I family endonuclease or a Type IV restriction endonuclease. 
     
     
         10 . A method according to  claim 1 , further comprising recording in a computer-readable form detection data indicative of detection of the hmN in (c). 
     
     
         11 . A kit for detecting a hydroxymethylated nucleotide (hmN) in a polynucleotide preparation; comprising:
 a glucosyltransferase and a site-specific endonuclease in a single container or in separate containers, the site-specific endonucleases being capable of cleaving a polynucleotide having a specific recognition site containing a methylated nucleotide (mN) or hmN but not a modified hmN (mhmN); and instructions for use in a method according to  claims 1  through  10 .   
     
     
         12 . A kit according to  claim 11 , further comprising a ligase and an adapter. 
     
     
         13 . A computer program product comprising:
 (a) a computer-readable storage medium; and   (b) instructions stored on the computer-readable storage medium that when executed by a computer cause the computer to:
 receive detection data obtained by the method according to  claim 10 ; 
 and perform at least one of: 
 (i) mapping the hmN detection data to a genome sequence; 
 (ii) comparing the detection data of hmNs in the polynucleotide preparation with a reference polynucleotide; and 
 (iii) identifying a biological marker from the detection data. 
   
     
     
         14 . A computer program product according to  claim 13 , wherein the instructions when executed by the computer further cause the computer to identify in the database a reference polynucleotide having substantially similar patterns of hydroxymethylation to that of the polynucleotide preparation. 
     
     
         15 . A computer program product according to  claim 13 , wherein the instructions when executed by the computer further cause the computer to search for a phenotype designation associated with the identified reference polynucleotide.