Analysis of methylation using nucleic acid arrays
First Claim
1. A method for determining the methylation status of a plurality of cytosines in a sample comprising genomic DNA, said method comprising:
- (a) fragmenting the sample with a first restriction enzyme that is insensitive to the methylation status of cytosines and a second restriction enzyme that is sensitive to the methylation status of cytosines to generate a first collection of fragments;
(b) adding a ligase to the first collection of fragments so that the ends of fragments are ligated together to form circular fragments, thereby generating a second collection of fragments;
(c) digesting linear fragments in the second collection of fragments;
(d) amplifying circular fragments in the second collection of fragments to generate a third collection of fragments;
(e) hybridizing the third collection of fragments to an array to obtain a hybridization pattern; and
(f) determining the methylation status of selected cytosines by analyzing the hybridization pattern.
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Abstract
Methods of analyzing DNA to determine the methylation status of a plurality of cytosines are disclosed. In one aspect genomic DNA is fragmented, fragments are circularized, the circles are treated with a methylation sensitive enzyme to enrich for circles with methylated sites or with a methylation dependent enzyme to enrich for circles with unmethylated sites, and the circles are amplified. The amplified product is fragmented, labeled and hybridized to an array of probes. The array of probes may be a tiling array or an array of junction probes. The hybridization pattern is analyzed to determine methylation status of cytosines.
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Citations
72 Claims
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1. A method for determining the methylation status of a plurality of cytosines in a sample comprising genomic DNA, said method comprising:
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(a) fragmenting the sample with a first restriction enzyme that is insensitive to the methylation status of cytosines and a second restriction enzyme that is sensitive to the methylation status of cytosines to generate a first collection of fragments;
(b) adding a ligase to the first collection of fragments so that the ends of fragments are ligated together to form circular fragments, thereby generating a second collection of fragments;
(c) digesting linear fragments in the second collection of fragments;
(d) amplifying circular fragments in the second collection of fragments to generate a third collection of fragments;
(e) hybridizing the third collection of fragments to an array to obtain a hybridization pattern; and
(f) determining the methylation status of selected cytosines by analyzing the hybridization pattern. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A method of classifying an unknown tumor into a known tumor class comprising:
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(a) obtaining a nucleic acid sample from said unknown tumor;
(b) fragmenting the nucleic acid sample with a first restriction enzyme that is insensitive to the methylation status of cytosines and a second restriction enzyme that is sensitive to the methylation status of cytosines to generate a first collection of fragments;
(c) adding a ligase to the first collection of fragments so that the ends of fragments in the first collection of fragments are ligated together to form circular fragments, thereby generating a second collection of fragments;
(d) amplifying circular fragments in the second collection of fragments and optionally digesting linear fragments, to generate a third collection of fragments;
(e) hybridizing the third collection of fragments to an array to obtain a hybridization pattern characteristic of said unknown tumor;
(f) obtaining a plurality of second hybridization patterns characteristic of each of a plurality of known tumor classes, wherein the second hybridization patterns are each generated according to the method of steps (a) to (e);
(g) comparing the first hybridization pattern characteristic of the unknown tumor to each of the second hybridization patterns to identify the second hybridization pattern that most closely matches the first hybridization pattern; and
(h) classifying the unknown tumor in the class of the tumor of known class with the most closely matched second hybridization pattern. - View Dependent Claims (27, 28, 29, 30, 31, 32)
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33. An array of probes comprising:
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at least 100,000 different probes comprising experimental probes and control probes, wherein at least 90% of the probes are experimental probes;
wherein each probe is present at a different, known or determinable, location in the array;
wherein at least 90% of the experimental probes are complementary to genomic target fragments, wherein a plurality of target fragments;
(a) are between 150 and 2000 base pairs when a selected mammalian genome is digested with a first restriction enzyme that recognizes a first recognition site; and
(b) comprise at least one second recognition site for a second restriction enzyme, wherein the second recognition site includes a CpG dinucleotide and said second restriction enzyme does not cleave at the second recognition site when the second recognition site is methylated. - View Dependent Claims (34, 35, 36, 37)
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38. A method of determining the presence or absence of methylation at a plurality of cytosines in a nucleic acid sample, comprising:
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(a) digesting the nucleic acid sample with a first restriction enzyme with a recognition site that includes a cytosine, wherein said first restriction enzyme is methylation sensitive;
(b) adding a ligase to the fragments generated in step (a) to generate circular fragments;
(c) digesting the products of step (b) with a restriction enzyme that is methylation dependent;
(d) digesting the products of step (c) to remove single stranded fragments;
(e) amplifying the products of step (d) using an amplification method that amplifies circular fragments;
(f) fragmenting and labeling the products of step (e);
(g) hybridizing the products of step (f) to an array of probes and detecting a resulting hybridization pattern; and
(h) analyzing the hybridization pattern to determine the methylation state of a plurality of cytosines in the nucleic acid sample. - View Dependent Claims (39, 40, 41, 42, 43, 44, 45, 46)
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47. A method of analyzing the methylation state of at least one cytosine in a nucleic acid sample, comprising:
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(a) fragmenting the nucleic acid sample to obtain a plurality of linear fragments;
(b) ligating a plurality of the fragments to form circular fragments;
(c) enriching the product of step (b) for methylated circular fragments by a method comprising;
mixing the circular fragments with one or more methylation sensitive restriction enzymes and an exonuclease;
(d) amplifying circular fragments from step (c) to generate an amplification product;
(e) fragmenting and labeling the amplification product;
(f) hybridizing the amplification product to an array of nucleic acid probes to obtain a hybridization pattern; and
,(g) analyzing the hybridization pattern to determine the methylation status of at least one cytosine. - View Dependent Claims (48, 49, 50)
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51. A method of obtaining a sample that is enriched for methylated sequences from a first sample, comprising:
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obtaining said first sample, wherein said first sample comprises methylated and unmethylated genomic DNA;
fragmenting said first sample using a restriction enzyme that is methylation insensitive to obtain a fragmented sample;
treating said fragmented sample with a ligase to circularize at least some of the fragments in the fragmented sample to generate a circularized sample;
treating said circularized sample with at least one methylation sensitive restriction enzyme to obtain a sample that is enriched for methylated sequences. - View Dependent Claims (52, 53, 54)
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55. A method of obtaining a sample that is enriched for unmethylated sequences from a first sample, comprising:
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obtaining said first sample, wherein said first sample comprises methylated and unmethylated genomic DNA;
fragmenting said first sample using a restriction enzyme that is methylation sensitive to obtain a fragmented sample;
circularizing at least some of the fragments in the fragmented sample to generate circular fragments;
generating linear fragments from circular fragments that contain a methylated recognition site for the methylation dependent restriction enzyme;
digesting a plurality of said linear fragments; and
,amplifying circular fragments to obtain a sample that is enriched for unmethylated sequences. - View Dependent Claims (56, 57, 58, 59, 60, 61, 62)
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63. A method for determining the methylation status of a plurality of cytosines in a sample comprising genomic DNA, said method comprising:
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(a) fragmenting the sample with a first restriction enzyme that is insensitive to the methylation status of cytosines and a second restriction enzyme that is sensitive to the methylation status of cytosines to generate a first collection of fragments;
(b) adding a ligase to the first collection of fragments so that the ends of fragments in the first collection of fragments are ligated together to form circular fragments, thereby generating a second collection of fragments;
(c) hybridizing a plurality of molecular inversion probes to the third collection of fragments wherein the molecular inversion probes include a 5′
region that is complementary to the first half of a predicted junction and a 3′
region that is complementary to the second half of a predicted junction so that when the predicted junction is present the molecular inversion probe can hybridize to the junction so that the 5′ and
3′
ends of the molecular inversion probe can be ligated together;
(d) ligating the ends of a plurality of the molecular inversion probes; and
(e) removing linear molecular inversion probes;
(f) amplifying the molecular inversion probes remaining after step (e); and
(g) detecting the presence of a plurality of molecular inversion probe amplicons in the amplified product of step (f), wherein the presence of a molecular inversion probe amplicon is indicative of the presence of a predicted junction; and
(h) determining the methylation status of selected cytosines. - View Dependent Claims (64, 65, 66)
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67. A computer implemented method for determining the methylation status of a plurality of cytosines in a sample comprising:
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providing a plurality of signals where in each signal represents the level of a junction in a sample;
providing a database of junction sequences;
comparing the plurality of signals to the database of junction sequences to identify a plurality of sequences that are present in the sample at a level above background; and
,analyzing the pattern of junctions to determine the methylation status of a plurality of cytosines. - View Dependent Claims (68)
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69. A computer software product comprising:
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computer program code that inputs a plurality of signals where each of said signals reflects the relative level of a junction in a sample, wherein junctions are formed by intramolecular ligation of restriction fragments;
computer program code that compares the relative level of a plurality of junctions to a database of junctions, wherein the formation of at least some of said junctions is dependent on the presence of methylation at a restriction site; and
,a computer readable media storing said computer program codes. - View Dependent Claims (70)
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71. A method of analyzing the methylation state of at least one cytosine in a nucleic acid sample, comprising:
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(a) fragmenting the nucleic acid sample to obtain a plurality of linear fragments;
(b) ligating a plurality of the fragments to form circular fragments;
(c) enriching the product of step (b) for unmethylated circular fragments by a method comprising;
mixing the circular fragments with one or more methylation dependent restriction enzymes and an exonuclease;
(d) amplifying circular fragments from step (c) to generate an amplification product;
(e) fragmenting and labeling the amplification product;
(f) hybridizing the amplification product to an array of nucleic acid probes to obtain a hybridization pattern; and
,(g) analyzing the hybridization pattern to determine the methylation status of at least one cytosine. - View Dependent Claims (72)
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Specification