Differential enzymatic fragmentation by whole genome amplification
First Claim
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1. A method of amplifying unmethylated or methylated DNA fragments in a biological sample, the method comprising:
- (a) providing randomly fragmented DNA from the biological sample;
(b) adding a sequence tag onto at least one end of the DNA fragments, thereby generating modified DNA fragments;
(c) digesting the modified DNA fragments with a methylation-dependent restriction enzyme or a methylation-sensitive restriction enzyme to obtain intact modified DNA fragments and digested modified DNA fragments; and
(d) after the digesting step, amplifying the intact modified DNA fragments with at least one primer that initiates amplification from the sequence tags, thereby generating amplified intact DNA fragments representing the unmethylated or methylated modified intact DNA fragments in the sample.
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Abstract
The present invention provides methods for detecting the presence of methylation at a locus within a population of nucleic acids.
58 Citations
38 Claims
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1. A method of amplifying unmethylated or methylated DNA fragments in a biological sample, the method comprising:
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(a) providing randomly fragmented DNA from the biological sample; (b) adding a sequence tag onto at least one end of the DNA fragments, thereby generating modified DNA fragments; (c) digesting the modified DNA fragments with a methylation-dependent restriction enzyme or a methylation-sensitive restriction enzyme to obtain intact modified DNA fragments and digested modified DNA fragments; and (d) after the digesting step, amplifying the intact modified DNA fragments with at least one primer that initiates amplification from the sequence tags, thereby generating amplified intact DNA fragments representing the unmethylated or methylated modified intact DNA fragments in the sample. - 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)
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24. A method for comparing the methylation state of a specific sequence in one portion of randomly fragmented DNA to the methylation state of the same sequence in at least a second portion of DNA, the method comprising:
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(a) providing a first and a second portion of DNA, wherein the first portion comprises randomly fragmented DNA; (b) in the first portion; (i) adding a sequence tag onto at least one end of the DNA fragments, thereby generating modified DNA fragments; (ii) digesting the DNA fragments with a methylation-dependent restriction enzyme, a methylation-sensitive restriction enzyme, a methylation-insensitive restriction enzyme, or a methylation-sensitive restriction enzyme and a methylation dependent restriction enzyme to obtain intact DNA fragments and digested DNA fragments; (iii) after the digesting step, amplifying the intact modified DNA fragments with at least one primer that initiates amplification from the sequence tags; (iv) quantifying the number of amplified intact modified DNA fragments comprising the specific sequence; and (c) comparing the number of amplified intact modified DNA fragments having the specific sequence in the first portion to the number of fragments having the specific sequence in the second portion, thereby determining an increase or decrease in methylation of a specific sequence. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38)
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Specification