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

• Current Assignee: Orion Genomics LLC
• Original Assignee: Orion Genomics LLC
• Inventors: Jeddeloh, Jeffrey A., Lakey, Nathan D.
• Primary Examiner(s): BAUSCH, SARAE L
• Application Number:
  US12/896,753
• Publication Number:
  US 20120100535A1
• Time in Patent Office: 851 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2545/101 with an internal standard/control

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.

Claims

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<sup>|Δ

Ct|</sup> is proportional to the relative methylation density.

2. A method for detecting methylation density in a locus of genomic DNA, the method comprising:

a. contacting a first portion of the genomic DNA containing the locus 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;

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.

3. The method of claim 2, further comprising:

contacting a third portion of the genomic DNA with a methylation-sensitive restriction enzyme and the methylation-dependent restriction enzyme to generate intact and cleaved copies of the locus;

quantifying the intact copies of the locus in the third portion following methylation-sensitive restriction enzyme and methylation-dependent restriction enzyme digestion.

4. The method of claim 3, further comprising:

contacting a fourth portion of the genomic DNA with a methylation-sensitive restriction enzyme to generate intact and cleaved copies of the locus;

quantifying the intact copies of the locus in the fourth portion following methylation-sensitive restriction enzyme digestion.

5. The method of claim 2, further comprising:

contacting a third portion of the genomic DNA with a methylation-sensitive restriction enzyme to generate intact and cleaved copies of the locus;

quantifying the intact copies of the locus in the third portion following methylation-sensitive restriction enzyme digestion.

6. The method of claim 2, wherein the quantifying steps comprise hybridizing two oligonucleotide primers to genomic DNA flanking the locus to produce an amplification product corresponding to the intact copies of the locus of genomic DNA between the primers.

7. The method of claim 6, wherein the quantifying steps comprise quantitative amplification of the intact copies of the locus.

8. The method of claim 3, wherein the quantifying steps comprise quantitative amplification of the intact copies of the locus.

9. The method of claim 4, wherein the quantifying steps comprise quantitative amplification of the intact copies of the locus.

10. The method of claim 5, wherein the quantifying steps comprise quantitative amplification of the intact copies of the locus.

11. The method of claim 7, wherein the quantitative amplification is real-time quantitative polymerase chain reaction (PCR).

12. The method of claim 7, wherein the method further comprises contacting the genomic DNA with an agent that modifies unmethylated cytosine before the amplification step, and at least one of the two oligonucleotide primers distinguishes between modified unmethylated and methylated DNA in the genomic DNA.

13. The method of claim 12, wherein the agent is sodium bisulfate.

14. The method of claim 2, wherein the quantifying steps comprise detecting a probe that hybridizes to the locus.

15. The method of claim 14, wherein the probe comprises a detectable fluorescent moiety.

16. The method of claim 2, wherein the genomic DNA is from an animal.

17. The method of claim 16, wherein the animal is a human.

18. The method of claim 2, wherein the genomic DNA is from an organism selected from the group consisting of plants, fungi and bacteria.

19. The method of claim 2, wherein the genomic DNA is from a tissue selected from the group consisting of brain tissue, colon tissue, urogenital tissue, lung tissue, renal tissue, breast tissue, thymus tissue, testis tissue, ovarian tissue, and uterine tissue.