Methods of analysis of methylation
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 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 with a methylation-sensitive restriction enzyme (MSRE) to obtain fragments, wherein said fragments comprise a mixture of target fragments and non-target fragments;
(c) filling in the ends generated by cleavage with the MSRE using a DNA polymerase;
(d) fragmenting the product of (c) with a methylation-insensitive restriction enzyme (MIRE) that recognizes the same restriction site as the MSRE;
(e) mixing the fragments of (d) 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;
(f) treating the products of (e) with an exonuclease to digest the template probes;
(g) treating the products of (f) with bisulfite;
(h) amplifying the products of (g) by PCR using primers to the common priming sequences to obtain an amplification product;
(i) hybridizing the amplification product from step (h) to an array of probes to obtain a hybridization pattern; and
(j) analyzing the hybridization pattern to detect the presence or absence of methylation at a plurality of cytosines in the target sequences.
2. The method of claim 1 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.
3. The method of claim 1 wherein the oligonucleotide complementary to said first common priming sequence comprises a plurality of phosphorothioate linkages.
4. The method of claim 1 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.
5. The method of claim 1 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.
6. The method of claim 1 wherein the common priming sequences contain 5-methylcytosine in place of cytosine.