Methods for analysis of nucleic acid methylation status and methods for fragmentation, labeling and immobilization of nucleic acids

Abstract

The invention relates to methods for analysis of nucleic acid methylation status, and fragmentation and/or labeling and/or immobilization of nucleic acids. More particularly, the invention relates to methods for fragmentation and/or labeling and/or immobilization of nucleic acids comprising labeling and/or cleavage and/or immobilization at abasic sites.

1 . A method for fragmenting a polynucleotide comprising a methylated nucleotide, said method comprising: 
 (a) cleaving a base portion of the methylated nucleotide with an agent capable of cleaving the base portion of the methylated nucleotide, whereby an abasic site is generated; and    (b) cleaving the phosphodiester backbone of the polynucleotide comprising the abasic site at the abasic site, whereby polynucleotide fragments are generated.    
     
     
         2 . A method according to  claim 1 , wherein the agent capable of cleaving the base portion of the methylated nucleotide is selected from the group consisting of an enzyme, a chemical agent, and acidic conditions.  
     
     
         3 . A method according to  claim 1 , wherein cleavage of the phosphodiester backbone is performed with an agent selected from the group consisting of an enzyme, a chemical agent, acidic conditions, basic conditions, and heat.  
     
     
         4 . A method for labeling a polynucleotide comprising a methylated nucleotide, said method comprising fragmenting the polynucleotide according to the method of  claim 1 , and further comprising: 
 (c) labeling at the abasic site, whereby a labeled polynucleotide fragment is generated.    
     
     
         5 . A method according to  claim 4 , wherein the labeled polynucleotide fragment comprises a detectable label.  
     
     
         6 . A method according to  claim 5 , further comprising hybridizing the labeled polynucleotide fragment to a probe.  
     
     
         7 . A method for labeling a polynucleotide comprising a methylated nucleotide, said method comprising: 
 (a) cleaving a base portion of the methylated nucleotide with an agent capable of cleaving the base portion of the methylated nucleotide, whereby an abasic site is generated; and    (b) labeling at the abasic site, whereby a labeled polynucleotide is generated;    
     
     
         8 . A method according to  claim 7 , further comprising hybridizing the labeled polynucleotide to a probe.  
     
     
         9 . A method for characterizing a methylated polynucleotide, said method comprising detecting a polynucleotide fragment, wherein said polynucleotide fragment is prepared according to the method of  claim 1 , wherein detection of said polynucleotide fragment correlates with presence, absence, sequence, or amount of the methylated polynucleotide.  
     
     
         10 . A method for characterizing a methylated polynucleotide, said method comprising detecting a labeled polynucleotide, wherein said labeled polynucleotide is prepared according to the method of  claim 7 , wherein detection of said labeled polynucleotide correlates with presence, absence, sequence, or amount of the methylated polynucleotide.  
     
     
         11 . A method for fragmenting a polynucleotide comprising a methylated nucleotide, said method comprising: 
 (a) cleaving a base portion of an unmethylated nucleotide with an agent capable of cleaving the base portion of the unmethylated nucleotide, whereby an abasic site is generated, wherein the agent is not capable of cleaving a methylated nucleotide;    (b) cleaving the phosphodiester backbone of the polynucleotide comprising the abasic site at the abasic site, whereby polynucleotide fragments are generated.    
     
     
         12 . A method according to  claim 11 , wherein the agent capable of cleaving a base portion of an unmethylated nucleotide comprises an enzyme.  
     
     
         13 . A method according to  claim 12 , wherein said the unmethylated nucleotide is cytosine and the enzyme comprises cytosine deaminase in conjunction with uracil DNA glycosylase.  
     
     
         14 . A method according to  claim 11 , wherein cleavage of the phosphodiester backbone is performed with an agent selected from the group consisting of an enzyme, a chemical agent, acidic conditions, basic conditions, and heat.  
     
     
         15 . A method according to  claim 11 , wherein the polynucleotide comprising a methylated nucleotide is contacted with a methyl binding agent prior to or during step (a).  
     
     
         16 . A method for labeling a polynucleotide comprising a methylated nucleotide, said method comprising fragmenting the polynucleotide according to the method of  claim 11 , and further comprising: 
 (c) labeling at the abasic site, whereby a labeled polynucleotide fragment is generated.    
     
     
         17 . A method according to  claim 16 , wherein the labeled polynucleotide fragment comprises a detectable label.  
     
     
         18 . A method according to  claim 17 , further comprising hybridizing the labeled polynucleotide fragment to a probe.  
     
     
         19 . A method for labeling a polynucleotide comprising a methylated nucleotide, said method comprising: 
 (a) cleaving a base portion of an unmethylated nucleotide with an agent capable of cleaving the base portion of the unmethylated nucleotide, whereby an abasic site is generated, wherein the agent is not capable of cleaving a methylated nucleotide; and    (b) labeling at the abasic site, whereby a labeled polynucleotide fragment is generated.    
     
     
         20 . A method according to  claim 19 , comprising hybridizing the labeled polynucleotide to a probe.  
     
     
         21 . A method for fragmenting and labeling a polynucleotide comprising a canonical nucleotide, said method comprising: 
 (a) cleaving a base portion of a canonical nucleotide present in a polynucleotide comprising the canonical nucleotide with an agent capable of cleaving a base portion of the canonical nucleotide, whereby an abasic site is generated;    (b) cleaving the phosphodiester backbone at the abasic site; and    (c) labeling at the abasic site, whereby a labeled polynucleotide fragment is generated.    
     
     
         22 . A method according to  claim 21 , wherein the agent capable of cleaving a base portion of a canonical nucleotide comprises an enzyme.  
     
     
         23 . A method according to  claim 22 , wherein said canonical nucleotide is cytosine and said enzyme comprises cytosine deaminase in conjunction with uracil DNA glycosylase.  
     
     
         24 . A method according to  21 , wherein cleavage of the phosphodiester backbone is performed with an agent selected from the group consisting of an enzyme, a chemical agent, acidic conditions, basic conditions, and heat.