Analysis of methylation status using oligonucleotide arrays
First Claim
1. A method for determining if a cytosine in a target sequence in a nucleic acid sample is methylated comprising:
- fragmenting the nucleic acid sample to generate fragments;
treating the sample with an agent that modifies unmethylated cytosines but does not modify methylated cytosines;
ligating an adaptor to the fragments, said adaptor comprising a first common sequence;
hybridizing a capture probe to the target sequence wherein the capture probe comprises a second common sequence, a tag sequence, a recognition sequence for a type IIs restriction enzyme and a region that is complementary to a region of the target sequence 3′
of the cytosine;
extending the capture probe to generate an extended capture probe;
amplifying the extended capture probe with first and second common sequence primers to generate double stranded extended capture probes;
digesting the amplified product with a Type IIS restriction enzyme to generate restriction fragments;
extending the restriction fragments in the presence of at least one labeled ddNTP;
hybridizing the restriction fragments to an array of oligonucleotides comprising a probe that is complementary to the tag sequence;
analyzing the hybridization pattern to determine the identity of labeled ddNTPs incorporated into the restriction fragments; and
determining the methylation status of the cytosine from the identity of labeled ddNTPs incorporated.
1 Assignment
0 Petitions
Accused Products
Abstract
The present invention provides for novel methods and kits for determining the methylation status of a cytosine in a nucleic acid sample. The methylation status of a plurality of cytosines may be determined simultaneously. In one embodiment methylation status is determined using methylation specific modification of cytosines followed by locus specific amplification, single base extension at the interrogation position and identification of the extended base by array hybridization. In another embodiment methylation specific modification of a cytosine is detected by hybridization to an array of probes that are perfectly complementary to either the methylated product of modification or the unmethylated product of modification. In another embodiment methylation status is determined using methylation specific restriction enzymes coupled with hybridization to an array.
-
Citations
39 Claims
-
1. A method for determining if a cytosine in a target sequence in a nucleic acid sample is methylated comprising:
-
fragmenting the nucleic acid sample to generate fragments;
treating the sample with an agent that modifies unmethylated cytosines but does not modify methylated cytosines;
ligating an adaptor to the fragments, said adaptor comprising a first common sequence;
hybridizing a capture probe to the target sequence wherein the capture probe comprises a second common sequence, a tag sequence, a recognition sequence for a type IIs restriction enzyme and a region that is complementary to a region of the target sequence 3′
of the cytosine;
extending the capture probe to generate an extended capture probe;
amplifying the extended capture probe with first and second common sequence primers to generate double stranded extended capture probes;
digesting the amplified product with a Type IIS restriction enzyme to generate restriction fragments;
extending the restriction fragments in the presence of at least one labeled ddNTP;
hybridizing the restriction fragments to an array of oligonucleotides comprising a probe that is complementary to the tag sequence;
analyzing the hybridization pattern to determine the identity of labeled ddNTPs incorporated into the restriction fragments; and
determining the methylation status of the cytosine from the identity of labeled ddNTPs incorporated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 27)
-
-
23. A method for determining the methylation status of at least one cytosine in each of a plurality of different target sequences in a nucleic acid sample comprising:
-
fragmenting the nucleic acid sample;
ligating an adaptor to the fragments, said adaptor comprising a first common sequence;
modifying unmethylated cytosines in the nucleic acid sample;
hybridizing the sample to a plurality of capture probes wherein each capture probe comprises a second common priming sequence, a common recognition sequence for a type IIS restriction enzyme, a tag sequence that is unique for each species of capture probe, and a region that hybridizes to a target sequence 3′
of a cytosine of interest and is unique for each species of capture probe;
extending the capture probes to generate an extended capture probes;
amplifying the extended capture probes with first and second common sequence primers;
digesting the amplified fragments with a Type IIS restriction enzyme to generate restriction fragments;
extending the restriction fragments in the presence of at least one labeled ddNTP;
hybridizing the restriction fragments to an array of oligonucleotides comprising probes that are complementary to the tag sequences; and
analyzing the hybridization pattern to determine the identity of labeled ddNTPs incorporated into the restriction fragments.
-
-
24. A method for determining the methylation status of a cytosine in a target sequence in a nucleic acid sample comprising:
-
fragmenting the nucleic acid sample to generate fragments;
differentially modifying methylated and unmethylated cytosines in the nucleic acid sample;
hybridizing a capture probe to the target sequence so that the 3′
end of the capture probe is adjacent to the cytosine and wherein the capture probe comprises a first common sequence, a tag sequence unique for each species of capture probe, and a region that hybridizes to the target sequence adjacent to the cytosine;
extending the capture probe to generate an extended capture probe;
hybridizing a target specific reverse primer to the extended capture probe wherein the locus specific reverse primer comprises a second common sequence and a target specific region that hybridizes to the target sequence 3′
of the cytosine and wherein either the capture probe or the target specific reverse primer comprises a recognition site for a type IIS restriction enzyme;
extending the target specific reverse primer to generate double stranded extended capture probe;
amplifying the double stranded extended capture probe with first and second common sequence primers;
digesting the amplified product with a Type IIS restriction enzyme to generate restriction fragments;
extending the restriction fragments in the presence of at least one labeled ddNTP;
hybridizing the restriction fragments to an array of oligonucleotides comprising a probe that is complementary to the tag sequence;
analyzing the hybridization pattern to determine the identity of labeled ddNTPs incorporated into the restriction fragments; and
determining the methylation status of the cytosine from the identify of labeled ddNTP incorporated. - View Dependent Claims (25, 26)
-
-
28. A kit for determining the methylation status of a cytosine present in a target sequence in a plurality of target sequences said kit comprising:
-
a collection of capture probes, wherein each species of capture probe comprises a first common sequence, a tag sequence unique for each species of capture probe, a first target specific sequence, a Type IIS restriction enzyme recognition sequence positioned to cleave immediately 5′
of a cytosine of interest, and a second target specific sequence;
an adaptor comprising a first strand comprising a second common sequence and a second strand that does not contain the complement of the second common sequence and is blocked from extension at the 3′
end; and
a pair of first and second common sequence primers.
-
-
29. A method of determining if a selected cytosine is methylated in a nucleic acid sample comprising;
-
in a first step, fragmenting the genomic DNA sample with a first enzyme;
in a second step, ligating an adaptor to the fragments to generate adaptor-ligated genomic fragments;
in a third step, dividing the sample into three portions; and
fragmenting the first portion with a first restriction enzyme that cleaves methylated DNA;
fragmenting the second portion with a second enzyme that is a methylation sensitive isoschizomer of the first enzyme; and
leaving the third portion of the sample untreated;
in a fourth step, amplifying each of the portions with a primer to the adaptor sequence;
in a fifth step separately hybridizing each of the amplified portions to an array of probes wherein the array interrogates the presence or absence of a plurality of sequences in the genomic sample; and
in a sixth step analyzing the hybridization patterns to determine presence or absence of a fragment in each portion wherein a fragment that is present in the second and third portions but not in the first portion indicates presence of methylated cytosine. - View Dependent Claims (30, 31, 32)
-
-
33. A method of determining the methylation status of a plurality of cytosines in a sample comprising:
-
fragmenting genomic DNA from the sample with a restriction enzyme;
modifying the fragments with sodium bisulfite;
ligating an adaptor sequence to the fragments;
amplifying at least a subset of the fragments;
labeling the amplified fragments;
hybridizing the fragments to an array of probes, wherein the array comprises a first set of probes comprises a plurality of probes that are each perfectly complementary to a subsequence of a target sequence wherein the subsequence comprises a cytosine to be interrogated for methylation and a second set of probes that corresponds to the first set of probes except that the positions that are complementary to cytosines in the target are changed to adenines. - View Dependent Claims (34, 35, 36, 37, 38, 39)
-
Specification