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Methods for quantitative determination of methylation density in a DNA locus

  • US 8,361,719 B2
  • Filed: 10/01/2010
  • Issued: 01/29/2013
  • Est. Priority Date: 10/21/2003
  • Status: Active Grant
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 a methylation-dependent restriction enzyme 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

    Ct|
    is proportional to the relative methylation density.

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