Methods of analysis of methylation

Abstract

Methods for determining the methylation status of a plurality of cytosines are disclosed. In some aspects genomic DNA target sequences containing CpGs are targeted for analysis by multiplex amplification using target specific probes that can be specifically degraded prior to amplification. The targets may be modified with bisulfite prior to amplification. In another aspect targets are cut with methylation sensitive or insensitive restriction enzymes and marked with a tag using the target specific probes. The presence or absence of methylation may be determined using methylation sensitive restriction enzyme or bisulfite treatment. Detection in many embodiments employs hybridization to tag arrays, genotyping arrays or resequencing arrays.

1 . A method for identifying sites of methylation in a genomic DNA sample said method comprising:
 (a) amplifying a plurality of target sequences by a method comprising:
 (i) hybridizing a plurality of first locus specific primers to the genomic DNA sample and extending said first locus specific primers, 
 (ii) hybridizing a plurality of second locus specific primers to the product of (i) and extending said second locus specific primers to obtain a plurality of templates comprising 5′ and 3′ ends defined by the first and second locus specific primers, 
 (iii) hybridizing a plurality of uracil containing probes to the product of (ii), wherein the uracil containing probes comprise a 5′ first common sequence, a 3′ second common sequence and a template complementary sequence, a first oligonucleotide that is complementary to the 5′ first common sequence and a second oligonucleotide that is complementary to the 3′ second common sequence, wherein the first and second oligonucleotides comprise methyl cytosine in place of cytosine, 
 (iv) ligating the first oligonucleotide to the 3′ end of the template and ligating the second oligonucleotide to the 5′ end of the template to generate a plurality of template fragments of known sequence with common sequences at the 5′ and 3′ ends; 
   (b) treat treating with uracil DNA glycosidase to fragment the uracil containing probes;   (c) treat treating with bisulfite to obtain bisulfite modified targets;   (d) amplify amplifying the bisulfite modified targets using primers to said first and second common sequences; and   (e) analyze analyzing cytosine positions in the target sequence for methylation by determining the sequence of the position in the amplified bisulfite modified targets obtained in step (d).   
     
     
         2 . The method of  claim 1 , wherein step (e) comprises: hybridization of the amplified target to a pair of sequence specific probes for each cytosine position to be analyzed wherein the pair of methylation specific probes comprises a first probe that is perfectly complementary to the cytosine position and surrounding bases with bisulfite modification blocked by methylation and a second probe that is perfectly complementary to the cytosine position and surrounding bases with bisulfite modification, wherein the presence or absence of methylation is determined by analyzing the pattern of hybridization of the amplified bisulfite modified target to the first and second probes. 
     
     
         3 . The method of  claim 1 , wherein sequence specific probes are attached to a solid support, selected from the group consisting of plurality of beads, one or more silica chip, one or more glass slides and one or more membranes, and wherein there are at least 1,000 pairs of sequence specific probes attached to said solid support. 
     
     
         4 - 13 . (canceled) 
     
     
         14 . A method of analyzing methylation status of a plurality of restriction sites comprising:
 (a) cutting the genomic DNA with a methylation dependent restriction enzyme (MDRE);   (b) end filling the restriction sites that were cut with the MDRE;   (c) cutting the product of (b) with a MSRE that is an isoschizomers of the MDRE;   (d) adding first and second dU probes to the products of (c) along with ligase, tag oligonucleotides and common sequence oligonucleotides;   (e) treating the products of (d) with UDG to fragment dU probes;   (f) amplifying the products of (e) by PCR with common primers; and   (g) detecting the tag sequences present in (f).   
     
     
         15 . The method of  claim 14 , wherein the MDRE is BisI and the MSRE is Fnu4HI. 
     
     
         16 - 19 . (canceled) 
     
     
         20 . A method for analyzing the methylation of a plurality of cytosines in a plurality of target sequences, said method comprising:
 (a) obtaining a genomic DNA sample;   (b) fragmenting the genomic DNA sample to obtain fragments, wherein said fragments comprise a mixture of target fragments and non-target fragments;   (c) mixing the fragments with (i) a plurality of template probes, each template probe comprising a target complementarity region, a first common priming sequence and a second common priming sequence, wherein said first common priming sequence is 3′ of the target complementarity region and said second common priming sequences is 5′ of the target complementarity region, (ii) an oligonucleotide complementary to said first common priming sequence and an oligonucleotide that is complementary to said second common priming sequence, wherein at least one of the oligonucleotides is exonuclease resistant, and (iii) a ligase, to obtain ligation products;   (d) treating the products of (c) with an exonuclease to digest the template probe;   (e) treating the products of (d) with bisulfite;   (f) amplifying the products of (e) by PCR using primers to the common primer sequences to obtain an amplification product;   (g) hybridizing the amplification product from step (g) to an array of probes to obtain a hybridization pattern; and   (h) analyzing the hybridization pattern to detect the presence or absence of methylation at a plurality of cytosines in the target sequences.   
     
     
         21 . The method of  claim 20 , wherein the exonuclease is a 5′ to 3′ exonuclease and the oligonucleotide complementary to said first common priming sequence is resistant to 5′ to 3′ exonuclease digestion. 
     
     
         22 . The method of  claim 20 , wherein the oligonucleotide complementary to said first common priming sequence comprises a plurality of phosphorothioate linkages. 
     
     
         23 . The method of  claim 20 , wherein the exonuclease is a 3′ to 5′ exonuclease and the oligonucleotide complementary to said second common priming sequence is resistant to 3′ to 5′ exonuclease digestion. 
     
     
         24 . The method of  claim 20 , wherein the array comprises a first plurality of probes that are perfectly complementary to a genomic region containing a CpG after bisulfite treatment and amplification if the region was unmethylated and a second plurality of probes that are perfectly complementary to the same region after bisulfite modification and amplification if the region was fully methylated. 
     
     
         25 . The method of  claim 20 , wherein the common primer sequences contain 5 methyl cytosine in place of cytosine.