Determination of methylated DNA
First Claim
1. A method of determining methylation of a nucleic acid molecule, comprising acts of:
- providing a nucleic acid molecule suspected of being methylated at a methylation site;
hybridizing a nucleic acid probe to the nucleic acid molecule proximate the methylation site to produce a nucleic acid molecule-nucleic acid probe hybrid;
exposing the nucleic acid-nucleic acid probe hybrid to a methyltransferase;
exposing the nucleic acid molecule-nucleic acid probe hybrid to a methylation-sensitive restriction endonuclease; and
determining a cleavage state of the nucleic acid probe to determine methylation of the nucleic acid at the methylation site.
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Accused Products
Abstract
The present invention generally relates to the determination of the state of one or more locations within a nucleic acid and, in particular, to the determination of the methylation state of one or more methylation sites within a nucleic acid such as DNA. In one aspect of the invention, a nucleic acid, such as DNA, that is suspected of being methylated is exposed to a nucleic acid probe able to hybridize the nucleic acid at or near the methylation site. After hybridization, the nucleic acid-probe hybrid is exposed to a methylation-sensitive restriction endonuclease able to bind at or near the methylation site. The restriction endonuclease is not able to cleave the nucleic acid-probe hybrid if the DNA is methylated at the methylation site, but is able to cleave the nucleic acid-probe hybrid if the nucleic acid is not methylated at the methylation site. Determination of the cleavage state of the probe can thus be used to determine the state of the methylation site.
87 Citations
26 Claims
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1. A method of determining methylation of a nucleic acid molecule, comprising acts of:
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providing a nucleic acid molecule suspected of being methylated at a methylation site; hybridizing a nucleic acid probe to the nucleic acid molecule proximate the methylation site to produce a nucleic acid molecule-nucleic acid probe hybrid; exposing the nucleic acid-nucleic acid probe hybrid to a methyltransferase; exposing the nucleic acid molecule-nucleic acid probe hybrid to a methylation-sensitive restriction endonuclease; and determining a cleavage state of the nucleic acid probe to determine methylation of the nucleic acid at the methylation site. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
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11. The method of claim IO, wherein the CpG island contained within the nucleic acid comprises the methylation site.
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24. A method of determining methylation of a nucleic acid molecule, comprising acts of:
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exposing a nucleic acid molecule to a surface having at least a first region comprising a first nucleic acid probe immobilized thereto and a second region comprising a second nucleic acid probe immobilized thereto, wherein the first nucleic acid probe is able to hybridize the nucleic acid molecule at a first region suspected of being methylated at a first methylation site, and the second nucleic acid probe is able to hybridize the nucleic acid molecule at a second region suspected of being methylated at a second methylation site different from the first methylation site; exposing at least one of the first nucleic acid probe and the second nucleic acid probe to a restriction endonuclease; and determining a cleavage state of the first nucleic acid probe and/or the second nucleic acid probe to determine, respectively, methylation of the nucleic acid at the first methylation site and/or the second methylation site.
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25. A method of determining the state of a target site of nucleic acid, comprising acts of:
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providing a nucleic acid molecule having a target site that can be in one of a plurality of naturally-occurring states, including a first state and a second state; hybridizing a nucleic acid probe to the nucleic acid molecule proximate the target site; exposing the nucleic acid-nucleic acid probe hybrid to a restriction endonuclease that does not bind the nucleic acid molecule if the target site is in a first state, but does bind the nucleic acid if the target site is in a second state; and thereafter, determining a cleavage state of the nucleic acid probe to determine the state of the target site.
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26. A kit for determining methylation of a nucleic acid molecule, the kit comprising:
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a nucleic acid probe comprising a hybridization region, a restriction site comprising a methylation site, and a detection entity; and a methylation-sensitive restriction endonuclease.
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Specification