Methods for quantitative determination of methylation density in a DNA locus
First Claim
Patent Images
1. A method for quantifying average methylation density in a locus of genomic DNA, the method comprising, contacting genomic DNA with a methylation-dependent restriction enzyme or a methylation-sensitive restriction enzyme under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved;
- quantifying intact copies of the locus; and
comparing the quantity of amplified product to a control value representing the quantity of methylation of control DNA, thereby quantifying the average methylation density in the locus compared to the methylation density of the control DNA.
1 Assignment
0 Petitions
Accused Products
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.
-
Citations
50 Claims
-
1. A method for quantifying average methylation density in a locus of genomic DNA, the method comprising,
contacting genomic DNA with a methylation-dependent restriction enzyme or a methylation-sensitive restriction enzyme under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved; -
quantifying intact copies of the locus; and
comparing the quantity of amplified product to a control value representing the quantity of methylation of control DNA, thereby quantifying the average methylation density in the locus compared to the methylation density of the control DNA. - 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, 26, 27, 28, 36, 37)
-
-
29. A method of calculating the relative methylation density for a target locus in a DNA sample, the method comprising,
i. contacting the DNA sample with a methylation-dependent restriction enzyme under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved to obtain a population of nucleic acids in which at least some methylated copies of the locus are fragmented, or contacting the DNA sample with a methylation-sensitive restriction enzyme under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved to obtain a population of nucleic acids in which at least some unmethylated copies of the locus are fragmented; -
ii. quantitatively amplifying intact copies of the locus in the DNA sample after the contacting steps;
iii. identifying the cycle-threshold (Ct) value for the amplified portion from the DNA sample; and
,iv. determining the relative methylation density for the target locus by calculating the difference (AΔ
Ct) between the Ct of the DNA sample and a control Ct value, wherein 2|Δ
Ct| is proportional to the relative methylation density between the DNA sample and the control. - View Dependent Claims (30, 31, 32, 33, 34, 35, 38)
-
-
39. A kit for quantifying the average methylation density in a locus of genomic DNA, the kit comprising
a methylation-dependent restriction enzyme or a methylation sensitive restriction enzyme; -
a control DNA molecule comprising a pre-determined number of methylated nucleotides; and
control oligonucleotide primers that hybridize to the control DNA molecule. - View Dependent Claims (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50)
-
Specification