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 with uracil DNA glycosidase to fragment the uracil containing probes;    (c) treat with bisulfite to obtain bisulfite modified targets;    (d) amplify the bisulfite modified targets using primers to said first and second common sequences; and    (e) analyze 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 . A method for determining the methylation status of a plurality of cytosines in a plurality of restriction sites for a methylation sensitive restriction enzyme (MSRE) in a genomic DNA sample comprising: 
 (a) fragmenting the genomic DNA sample with the MSRE;    (b) filling the ends generated by cleavage with the MSRE using a DNA polymerase;    (c) fragmenting the product of (b) with a methylation insensitive restriction enzyme (MIRE) that recognizes the same restriction site;    (d) hybridize the products of (c) with methylation specific dU probes, wherein each comprises a barcode tag sequence, a 5′ first common priming sequence and a 3′ second common priming sequence;    (e) add a ligase and oligonucleotides that are complementary to the barcode tag sequences, the first common priming sequence and the second common priming sequence under conditions to allow ligation of the target to the oligonucleotides;    (f) digest the products of (e) with uracil DNA glycosidase;    (g) amplify the product of (f) by PCR using primers to the first and second common priming sequence; and,    (h) detect the presence or absence of amplified barcode tag sequences from (g) by hybridization to determine methylation status of selected cytosines.    
     
     
         5 . The method of  claim 4  wherein for each restriction site being interrogated there is a first dU probe and a second dU probe, wherein the first dU probe is complementary to the fragment generated if the MSRE cut at the restriction site and the second dU probe is complementary to the fragment generated if the MSRE did not cut at the restriction site and wherein each dU probe has a different barcode tag sequence.  
     
     
         6 . The method of  claim 5  wherein the presence of the barcode sequence of the first dU probe indicates that the restriction site was unmethylated in at least some fragments and presence of the barcode tag sequence of the second dU probe indicates that the restriction site was methylated in at least some fragments.  
     
     
         7 . The method of  claim 4  wherein the barcode tag sequences are detected by hybridization to an array of barcode tag complements in known or determinable positions of the array.  
     
     
         8 . The method of  claim 7  wherein the array comprises more than 10,000 different barcode tag complements.  
     
     
         9 . The method of  claim 4  wherein the barcode tag sequence is between 20 and 30 bases in length.  
     
     
         10 . The method of  claim 4  wherein the DNA polymerase is Klenow.  
     
     
         11 . The method of  claim 4  wherein at least 500 different restriction sites are analyzed.  
     
     
         12 . The method of  claim 4  wherein the MSRE is Hpa II or Sma I and the MIRE is Msp I or Xma I.  
     
     
         13 . The method of  claim 7  wherein the barcode tag complements are attached to a solid support selected from the group consisting of beads, chips, and membranes.  
     
     
         14 . A method of analyzing methylation status of a plurality of restriction sites comprising: 
 (a) cutting the genomic DNA with a 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 . A method of detecting the presence or absence of methylation at a restriction site in a genomic DNA sample, said method comprising: 
 (a) treating said genomic DNA sample with a methylation sensitive restriction enzyme;    (b) treating the products of (a) with Klenow;    (c) treating the products of (b) with a methylation insensitive isoschizomers of the methylation sensitive restriction enzyme used in (a);    (d) adding to the products of (c) the following nucleic acids:    (i) a first dU probe and a second dU probe wherein said first dU probe is complementary to a strand of genomic DNA immediately adjacent to the restriction site and including the region filled in by Klenow and said second dU probe is complementary to the same strand of genomic DNA but does not include the region filled in by Klenow and wherein said first and second dU probes further comprise first and second common priming regions wherein said first dU probe has a first tag complement region and said second dU probe has a second tag complement region,    (ii) a first tag oligonucleotide that is complementary to said first tag complement region and a second tag complement that is complementary to said second tag complement;    (iii) oligonucleotides complementary to the first and second common priming regions; and    (iv) ligase;    (d) incubating to allow ligation;    (e) adding a UDG activity to the product of (d);    (f) amplifying the product of (e) by PCR using primers to the common priming regions; and    (g) detecting the presence of said first tag sequence or said second tag sequence, wherein presence of said first tag sequence indicates that the restriction site was unmethylated and presence of said second tag sequence indicates that the restriction site was methylated.    
     
     
         17 . The method of  claim 16  wherein said step of detecting comprises hybridization to an array of probes complementary to tag sequences.  
     
     
         18 . A method of detecting the presence or absence of methylation at a plurality of restriction sites according to the method of  claim 16 .  
     
     
         19 . The method of  claim 18  wherein the plurality of restriction sites includes at least 200 different restriction sites.  
     
     
         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 probes;    (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.