Methods for quantitative determination of methylation density in a DNA locus

• US 20050153316A1
• Filed: 10/21/2004
• Published: 07/14/2005
• Est. Priority Date: 10/21/2003
• Status: Active Grant

• Current Assignee: Orion Genomics LLC
• Original Assignee: Orion Genomics LLC
• Inventors: Lakey, Nathan D., Jeddeloh, Jeffrey A.
• Granted Patent: US 7,910,296 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 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.

2. The method of claim 1, wherein the quantifying step comprises a quantitative amplification.

3. The method of claim 2, wherein the quantity of the amplified product is compared to a standard curve.

4. The method of claim 1, wherein the amplifying step comprises hybridizing two oligonucleotide primers to DNA flanking the locus to produce an amplification product corresponding to the uncleaved locus of genomic DNA between the primers.

5. The method of claim 1, wherein the control value represents the quantity of an amplification product of a DNA sample having a known or predicted number of methylated nucleotides.

6. The method of claim 1, wherein the restriction enzyme is a methylation-sensitive restriction enzyme.

7. The method of claim 1, wherein the methylation-sensitive restriction enzyme is selected from the group consisting of Aat II, Aci I, Acl I, Age I, Alu I, Asc I, Ase L AsiS I, Bbe I, BsaA I, BsaH I, BsiE I, BsiW I, BsrF I, BssH II, BssK I, BstB I, BstN I, BstU I, Cla I, Eae I, Eag I, Fau I, Fse I, Hha I, HinP1 I, HinC II, Hpa II, Hpy99 I, HpyCH4 IV, Kas I, Mlu I, MapA1 I, Msp I, Nae I, Nar I, Not I, Pml I, Pst I, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, Sfl I, Sfo I, SgrA I, Sma I, SnaB I, Tsc I, Xma I, and Zra I.

8. The method of claim 1, wherein the restriction enzyme is a methylation-dependent restriction enzyme.

9. The method of claim 1, wherein the restriction enzyme is a methyl-cytosine-dependent restriction enzyme.

10. The method of claim 9, wherein the restriction enzyme is McrBC, McrA, or MrrA.

11. The method of claim 1, wherein the restriction enzyme is a methyl-adenosine-dependent restriction enzyme.

12. The method of claim 11, wherein the restriction enzyme is DpnI.

13. The method of claim 1, wherein the methylation-sensitive or methylation dependent restriction enzyme is contacted to the portion under conditions to allow for only a partial digest of the portion.

14. The method of claim 1, comprising

separating the genomic DNA into at least two equal portions;

contacting one portion with a methylation-sensitive or methylation dependent restriction enzyme and contacting a second portion with the isoschizomeric partner of the restriction enzyme,

amplifying the locus of genomic DNA in each portion in a step comprising hybridizing two oligonucleotide primers to DNA flanking the locus;

quantifying the amplification product; and

comparing the quantity of amplified products from the two portions.

15. The method of claim 1, wherein

the method further comprises contacting the genomic DNA with an agent that modifies unmethylated cytosine before the amplifying step, and

at least one of the two oligonucleotide primers distinguishes between modified unmethylated and methylated DNA in the genomic DNA.

16. The method of claim 15, further comprising contacting the DNA with at least one methylation-sensitive restriction enzyme or methylation-sensitive restriction enzyme before the genomic DNA is contacted with an agent that modifies unmethylated cytosine.

17. The method of claim 16, wherein the genomic DNA is contacted with a mixture of at least two different methylation-sensitive or methylation-dependent restriction enzymes.

18. The method of claim 15, wherein the agent is sodium bisulfite.

19. The method of claim 2, wherein the amplified product is quantified using quantitative PCR.

20. The method of claim 1, wherein the control value is generated by contacting DNA comprising a control locus with a methylation-dependent or methylation-sensitive restriction enzyme;

amplifying the control locus; and

determining the quantity of the amplified product.

21. The method of claim 20, wherein the control locus is known or predicted to be unmethylated.

22. The method of claim 1, wherein the control value comprises a known number of methylated nucleotides.

23. The method of claim 1, wherein the method is performed to detect the presence or absence of cancer cells in a subject.

24. The method of claim 1, wherein the quantifying step comprises detecting a probe that hybridizes to the amplification product.

25. The method of claim 24, wherein the probe comprises a detectable fluorescent moiety.

26. The method of claim 1, wherein the DNA is from an animal.

27. The method of claim 26, wherein the animal is a human.

28. The method of claim 1, wherein the genomic DNA is from an organism selected from the group consisting of plants, fungi and bacteria.

36. The method of claim 1, wherein the restriction enzyme is a methyl-adenosine-dependent restriction enzyme.

37. The method of claim 36, wherein the restriction enzyme is Dpn I.

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<sup>|Δ

Ct|</sup> is proportional to the relative methylation density between the DNA sample and the control.

30. The method of claim 29, wherein the control Ct is calculated by steps comprising:

i. contacting a control 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 control 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. amplifying intact copies of the locus in the control DNA sample after the contacting steps; and

,

iii. identifying the cycle-threshold (Ct) value for the amplified portion from the control DNA sample.

31. The method of claim 29, wherein the amplifying step comprises hybridizing two oligonucleotide primers to DNA flanking the locus to produce an amplification product corresponding to the uncleaved locus of genomic DNA between the primers.

32. The method of claim 29, wherein the methylation-sensitive restriction enzyme is selected from the group consisting of Aat II, Acl I, Age I, Alu I, Asc I, Ase I, AsiS I, Bbe I, BsaA I, BsaH I, BsiE I, BsiW I, BsrF I, BssH II, BssK I, BstB I, BstN I, BstU I, Cla I, Eae I, Eag I, Fau I, Fse I, Hha I, HinP1 II, HinC II, Hpa II, Hpy99 I, HpyCH4 IV, Kas I, Mlu I, MapAl I, Msp I, Nae I, Nar I, Not I, Pml I, Pst I, Pvu I, Rsr II, Sac II, Sap I, Sau3A I, Sfl I, Sfo I, SgrA I, Sma I, SnaB I, Tsc I, Xma I, and Zra I.

33. The method of claim 29, wherein the restriction enzyme is a methylation-dependent restriction enzyme.

34. The method of claim 29, wherein the restriction enzyme is a methyl-cytosine-dependent restriction enzyme.

35. The method of claim 34, wherein the restriction enzyme is McrBC, McrA, or MrrA.

38. The method of claim 29, wherein the methylation-sensitive or methylation dependent restriction enzyme is contacted to the portion under conditions to allow for only a partial digest of the portion.

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.

40. The kit of claim 39, wherein the restriction enzyme is a methylation-sensitive restriction enzyme.

41. The kit of claim 39, wherein the restriction enzyme is a methylation-dependent restriction enzyme.

42. The kit of claim 39, wherein the restriction enzyme is a methyl-cytosine-dependent restriction enzyme.

43. The kit of claim 39, wherein the restriction enzyme is McrBC, McrA, or MrrA.

44. The kit of claim 39, further comprising target oligonucleotide primers that hybridize to a pre-determined locus of genomic DNA.

45. The kit of claim 39, wherein at least one target oligonucleotide primer distinguishes between modified unmethylated and methylated DNA in human genomic DNA.

46. The kit of claim 39, wherein the kit comprises a plurality of DNA molecules comprising different pre-determined numbers of methylated nucleotides.

47. The kit of claim 39, further comprising reagents sufficient to support the activity of the restriction enzyme.

48. The kit of claim 39, further comprising a thermostable DNA polymerase.

49. The kit of claim 39, further comprising an agent that modifies unmethylated cytosine.

50. The kit of claim 39, further comprising a detectably-labeled oligonucleotide.