Methods of analysis of methylation
AssigneeAffymetrix, Inc.
InventorsShivani Nautiyal, Malek Faham
Life of this patent
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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.
Claims13
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 in 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 as the MSRE;
- (d) hybridizing the products of (c) with methylation-specific dU probes, wherein each dU probe comprises a barcode tag sequence, a 5′ first common priming sequence and a 3′ second common priming sequence;
- (e) adding 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 oligonucleotides to a product of (c) hybridized to a dU probe;
- (f) digesting the products of (e) with uracil DNA glycosylase;
- (g) amplifying the product of (f) by PCR using primers to the first and second common priming sequences;
- and,
- (h) detecting the presence or absence of amplified barcode tag sequences from (g) by hybridization to determine the methylation status of said plurality of cytosines.
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 DNA polymerase to fill in the ends generated by cleavage with the methylation-sensitive restriction enzyme;
- (c) treating the products of (b) with a methylation-insensitive isoschizomer 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, wherein said first and second dU probes further comprise first and second common priming regions, and 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 oligonucleotide that is complementary to said second tag complement;
- (iii) oligonucleotides complementary to the first and second common priming regions;
- and
- (iv) ligase;
- (e) incubating to allow ligation of the nucleic acids set forth in (ii) and (iii) to a product of (c) hybridized to a dU probe;
- (f) adding uracil DNA glycosylase to the product of (e);
- (g) amplifying the product of (f) by PCR using primers to the common priming regions;
- and
- (h) detecting the presence of said first tag sequence or said second tag sequence, wherein the presence of said first tag sequence indicates that the restriction site was unmethylated and the presence of said second tag sequence indicates that the restriction site was methylated.
Dependencies are inferred from the claim text.