Methods of copying the methylation pattern of DNA during isothermal amplification and microarrays

• US 20060257905A1
• Filed: 04/11/2006
• Published: 11/16/2006
• Est. Priority Date: 04/14/2005
• Status: Active Grant

• Current Assignee: Euclid Diagnostics Lcc, Euclid Diagnostics LLC
• Original Assignee: Euclid Diagnostics LLC
• Inventors: Freije, Wadiha, Brikun, Igor
• Granted Patent: US 7,449,297 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

A method for copying the methylation patterns of molecules of genomic DNA (MGD) during isothermal amplification of the MGD comprising obtaining MGD, copying the methylation patterns of the MGD using a DNA methylation-maintenance enzyme, while isothermally amplifying the MGD using a DNA polymerase with strand displacement activity, under conditions that simultaneously promote activity of the DNA methylation-maintenance enzyme and the DNA polymerase; a method for copying the methylation patterns in double-stranded DNA molecules during isothermal amplification of the DNA molecules comprising obtaining DNA molecules, contacting the DNA molecules with transposable elements and an enzyme, which can randomly insert the transposable elements into the DNA molecules, copying the methylation patterns of the DNA molecules using a DNA methylation-maintenance enzyme, while isothermally amplifying the DNA molecules using a DNA polymerase with strand displacement activity, under conditions that simultaneously promote activity of the DNA methylation-maintenance enzyme and the DNA polymerase; a buffer comprising a divalent ion, deoxynucleotide triphosphates (dNTPs), primers, and S-adenosyl-methionine (SAM); and a composition comprising a DNA methylation-maintenance enzyme, a DNA polymerase with strand displacement activity, and the buffer.

Claims

1. A method for copying the methylation patterns of molecules of genomic DNA (MGD) during isothermal amplification of the MGD, which method comprises:

(i) obtaining MGD;

(ii) copying the methylation patterns of the MGD using a DNA methylation-maintenance enzyme, while isothermally amplifying the MGD using a DNA polymerase with strand displacement activity, under conditions that simultaneously promote activity of the DNA methylation-maintenance enzyme and the DNA polymerase with strand displacement activity; and

, optionally,

(iii) purifying the MGD as necessary to enable further manipulation;

whereupon the MGD are amplified and their methylation patterns are copied.

2. The method of claim 1, wherein the conditions that simultaneously promote activity of the DNA methylation-maintenance enzyme and the DNA polymerase with strand displacement activity comprise the presence of a buffer comprising a divalent ion, deoxynucleotide triphosphates (dNTPs), primers, and S-adenosyl-methionine (SAM).

3. The method of claim 1, wherein step (ii) further comprises:

(i′

) mixing the MGD and primers, wherein either of (a) the MGD or (b) the MGD and the primers are optionally denatured before or after mixing,

(ii′

) incubating the mixture of (i′

) at a temperature from at least about 0°

C. to about 75°

C. for a sufficient amount of time to allow annealing of the primers to the MGD,

(iii′

) equilibrating the mixture of (ii′

) to an optimal temperature for methyltransferase activity,

(iv′

) adding a buffer, which comprises a divalent ion, dNTPs, primers, and SAM, and which is already equilibrated to the optimal temperature of (iii′

), and simultaneously with or sequentially to adding the buffer, adding the DNA methylation-maintenance enzyme and the DNA polymerase with strand displacement activity to the mixture of (iii′

), and

(v′

) incubating the mixture of (iv′

) at an optimal temperature for methyltransferase activity for a sufficient amount of time to allow methylation-coupled amplification of the MGD until a desired amount is obtained.

4. The method of claim 1, wherein the MGD are obtained from circulating DNA.

5. The method of claim 1, wherein the methylation-maintenance enzyme is from a eukaryote.

6. The method of claim 5, wherein the eukaryote is an Annelida.

7. The method of claim 6, wherein the Annelida is a Polychaeta.

8. The method of claim 7, wherein the Polychaeta is a marine annelid worm.

9. The method of claim 8, wherein the marine annelid worm is Chaetopterus variopedatus.

10. The method of claim 5, wherein the eukaryote is a Clitellata.

11. The method of claim 10, wherein the Clitellata is an earthworm.

12. The method of claim 5, wherein the eukaryote is a fungus.

13. The method of claim 12, wherein the fungus is Neurospora crassa.

14. The method of claim 5, wherein the eukaryote is a human.

15. The method of claim 5, wherein the eukaryote is a mouse.

16. The method of claim 1, wherein the methylation-maintenance enzyme is DNA methyltransferase-1 (DNMT-1), which is optionally modified to reduce or eliminate de novo methylation activity.

17. The method of claim 1, wherein the MGD are completely or partially double-stranded, in which case the method optionally further comprises contacting the MGD with transposable elements and an enzyme, which can randomly insert the transposable elements into the MGD, whereupon the MGD comprise the randomly inserted transposable elements.

18. The method of claim 17, wherein the method further comprises denaturing the MGD prior to or during step (ii).

19. The method of claim 1, wherein the MGD are single-stranded and comprised of fragments.

20. The method of claim 19, which further comprises contacting the MGD with an enzyme that ligates single-stranded nucleic acids under reaction conditions that promote ligation, optionally in the presence of a polynucleotide kinase.

21. The method of claim 1, wherein the MGD comprise fragments having shorter strands and longer strands.

22. The method of claim 21, which further comprises contacting the MGD with an enzyme that generates blunt ends or compatible ends on the fragments under reaction conditions that promote the generation of such ends.

23. The method of claim 22, wherein the blunt ends are generated by extending the shorter strands of the fragments, by shortening the longer strands of the fragments, or a combination thereof, optionally in the presence of a polynucleotide kinase.

24. The method of claim 22, which method further comprises contacting the MGD with an enzyme that ligates double-stranded nucleic acids under reaction conditions that promote ligation, optionally in the presence of a polynucleotide kinase.

25. The method of claim 23, which method further comprises contacting the MGD with an enzyme that ligates double-stranded nucleic acids under reaction conditions that promote ligation, optionally in the presence of a polynucleotide kinase.

26. A method for copying the methylation patterns of double-stranded DNA molecules during isothermal amplification of the DNA molecules, which method comprises:

(i) obtaining DNA molecules;

(ii) contacting the DNA molecules with transposable elements and an enzyme, which can randomly insert the transposable elements into the DNA molecules, whereupon the DNA molecules comprise the randomly inserted transposable elements;

(iii) copying the methylation patterns of the DNA molecules using a DNA methylation-maintenance enzyme, while isothermally amplifying the DNA molecules using a DNA polymerase with strand displacement activity, under conditions that simultaneously promote activity of the DNA methylation-maintenance enzyme and the DNA polymerase with strand displacement activity, and, optionally;

(iv) purifying the DNA molecules as necessary to enable further manipulation;

whereupon the DNA molecules are amplified and their methylation patterns are copied.

27. The method of claim 26, wherein the conditions that simultaneously promote activity of the DNA methylation-maintenance enzyme and the DNA polymerase with strand displacement activity comprise the presence of a buffer comprising a divalent ion, dNTPs, primers, and SAM.

28. The method of claim 26, wherein step (iii) further comprises:

(i′

) mixing the DNA molecules and the primers, wherein either of (a) the DNA molecules or (b) the DNA molecules and the primers are optionally denatured before or after mixing,

(ii′

) incubating the mixture of (i′

) at a temperature from at least about 10°

C. to about 75°

C. for a sufficient amount of time to allow annealing of the primers to the DNA molecules,

(iii′

) equilibrating the mixture of (ii′

) to an optimal temperature for methyltransferase activity,

(iv′

) adding a buffer, which comprises a divalent ion, dNTPs, primers, and SAM, and which is already equilibrated to the optimal temperature of (iii′

), and simultaneously with or sequentially to adding the buffer, adding the DNA methylation-maintenance enzyme and the DNA polymerase with strand displacement activity to the mixture of (iii′

), and

(v′

) incubating the mixture of (iv′

) at an optimal temperature for methyltransferase activity for a sufficient amount of time to allow methylation-coupled amplification of the DNA molecules until a desired amount is obtained.

29. The method of claim 26, wherein the DNA molecules are obtained from circulating DNA.

30. The method of claim 26, wherein the methylation-maintenance enzyme is from a eukaryote.

31. The method of claim 30, wherein the eukaryote is an Annelida.

32. The method of claim 31, wherein the Annelida is a Polychaeta.

33. The method of claim 32, wherein the Polychaeta is a marine annelid worm.

34. The method of claim 33, wherein the marine annelid worm is Chaetopterus variopedatus.

35. The method of claim 31, wherein the Annelida is a Clitellata.

36. The method of claim 35, wherein the Clitellata is an earthworm.

37. The method of claim 30, wherein the eukaryote is a fungus.

38. The method of claim 37, wherein the fungus is Neurospora crassa.

39. The method of claim 30, wherein the eukaryote is a human.

40. The method of claim 30, wherein the eukaryote is a mouse.

41. The method of claim 26, wherein the methylation-maintenance enzyme is DNMT-1, which is optionally modified to reduce or eliminate de novo methylation activity.

42. The method of claim 26, wherein the method further comprises denaturing the DNA molecules prior to or during step (iii).

43. The method of claim 26, wherein the DNA molecules comprise fragments having shorter strands and longer strands.

44. The method of claim 26, which further comprises contacting the DNA molecules with an enzyme that generates blunt ends or compatible ends on the fragments under reaction conditions that promote the generation of such ends.

45. The method of claim 44, wherein the blunt ends are generated by extending the shorter strands of the fragments, by shortening the longer strands of the fragments, or a combination thereof, optionally in the presence of a polynucleotide kinase.

46. The method of claim 44, which method further comprises contacting the DNA molecules with an enzyme that ligates double-stranded nucleic acids under reaction conditions that promote ligation, optionally in the presence of a polynucleotide kinase.

47. The method of claim 45, which method further comprises contacting the DNA molecules with an enzyme that ligates double-stranded nucleic acids under reaction conditions that promote ligation, optionally in the presence of a polynucleotide kinase.

48. A buffer comprising a divalent ion, dNTPs, primers, and SAM.

49. A composition for methylation-coupled amplification of DNA comprising a DNA methylation-maintenance enzyme, a DNA polymerase with strand displacement activity, and the buffer of claim 48.