Methods for quantitative determination of methylation density in a DNA locus
First Claim
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1. A method of calculating the relative methylation density for a target locus in a DNA sample, the method comprising,i. contacting a first portion of the DNA sample with McrBC under partial digestion conditions where the resulting number of remaining intact copies of the locus is inversely proportional to the quantity of methylation at the locus;
- ii. quantitatively amplifying intact copies of the locus in the DNA sample after step i;
iii. identifying a cycle-threshold (Ct) value from step ii;
iv. quantitatively amplifying intact copies of the locus in a second portion of the genomic DNA that has not been cut by a restriction enzyme;
v. identifying a cycle-threshold (Ct) value from step iv; and
,vi. determining the relative methylation density for the target locus by calculating the difference (Δ
Ct) between the Ct from step iii and the Ct from step v, wherein 2|Δ
Ct| is proportional to the relative methylation density.
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Abstract
The present invention is a novel method of determining the average DNA methylation density of a locus of interest within a population of DNA fragments.
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19 Claims
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1. A method of calculating the relative methylation density for a target locus in a DNA sample, the method comprising,
i. contacting a first portion of the DNA sample with McrBC under partial digestion conditions where the resulting number of remaining intact copies of the locus is inversely proportional to the quantity of methylation at the locus; -
ii. quantitatively amplifying intact copies of the locus in the DNA sample after step i; iii. identifying a cycle-threshold (Ct) value from step ii; iv. quantitatively amplifying intact copies of the locus in a second portion of the genomic DNA that has not been cut by a restriction enzyme; v. identifying a cycle-threshold (Ct) value from step iv; and
,vi. determining the relative methylation density for the target locus by calculating the difference (Δ
Ct) between the Ct from step iii and the Ct from step v, wherein 2|Δ
Ct| is proportional to the relative methylation density.
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2. A method for detecting methylation density in a locus of genomic DNA, the method comprising:
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a. contacting a first portion of the genomic DNA containing the locus with McrBC under partial digestion conditions where the resulting number of remaining intact copies of the locus is inversely proportional to the quantity of methylation at the locus; b. quantifying the remaining intact copies of the locus in the first portion following step a; c. quantifying intact copies of the locus in a second portion of the genomic DNA that has not been cut by a restriction enzyme, wherein comparison of the quantity of intact copies of the locus in step b to the quantity of intact copies of the locus in step c allows for methylation density detection. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification