Methods and compositions for generating and amplifying DNA libraries for sensitive detection and analysis of DNA methylation

• US 20050202490A1
• Filed: 03/03/2005
• Published: 09/15/2005
• Est. Priority Date: 03/08/2004
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Rubicon Genomics, Inc (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Tarrier, Brendan J., Tesmer, Tim, M'Mwirichia, Joseph, Bruening, Eric E., Sun, Tong, Kurihara, Takao, Makarov, Vladimir L., Kamberov, Emmanuel, Pinter, Jonathon H.
• Granted Patent: US 8,440,404 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

The present invention regards a variety of methods and compositions for obtaining epigenetic information, such as DNA methylation patterns, through the preparation, amplification and analysis of Methylome libraries. In several aspects of the present invention, there are methods based on methylation-dependent enrichment or depletion of genomic DNA isolated from cellular and cell-free sources. In additional embodiments, there are methods and compositions for single-step high throughput preparations of Methylome libraries.

Claims

1. A method of preparing a DNA molecule, comprising:

(a) providing a DNA molecule;

(b) digesting the DNA molecule with at least one methylation-sensitive restriction enzyme and, optionally, an additional nuclease, to provide digested DNA molecules;

(c) incorporating a nucleic acid molecule into at least some of the digested DNA molecules to provide first modified DNA molecules, by one of the following;

(1) incorporating at least one primer from a plurality of primers, said primers comprising a 5′

constant sequence and a 3′

variable sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality;

or

(2) incorporating an oligonucleotide comprising an inverted repeat and a loop, under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the molecule, thereby producing an oligonucleotide-linked molecule comprising a nick having a 3′

hydroxyl group, wherein there is polymerization from the 3′

hydroxyl group of at least part of the oligonucleotide-linked molecule; and

(d) amplifying one or more of the first modified DNA molecules to provide amplified first modified DNA molecules.

2. The method of claim 1, wherein the incorporating step comprises incorporating at least one primer from a plurality of primers, said primers comprising a 5′

constant sequence and a 3′

variable sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality.

3. The method of claim 1, wherein the incorporating step comprises incorporating an oligonucleotide comprising an inverted repeat and a loop, under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the molecule, thereby producing an oligonucleotide-linked molecule comprising a nick having a 3′

hydroxyl group, wherein there is polymerization from the 3′

hydroxyl group of at least part of the oligonucleotide-linked molecule.

4. The method of claim 1, further comprising analyzing at least part of the sequence of an amplified modified DNA molecule.

5. The method of claim 1, wherein the DNA molecule that is provided is from a body fluid or tissue.

6. The method of claim 5, wherein the body fluid comprises blood, serum, urine, cerebrospinal fluid, nipple aspirate, sweat, or saliva.

7. The method of claim 5, wherein the tissue comprises biopsy, surgical sample, or cheek scrapings.

8. The method of claim 1, wherein the DNA molecule that is provided is from a sample of an individual that has a medical condition.

9. The method of claim 8, wherein the medical condition is cancer.

10. The method of claim 1, wherein the methylation-sensitive restriction enzyme has a 4-5 base pair recognition site that comprises at least one CpG dinucleotide.

11. The method of claim 1, wherein the methylation-sensitive restriction enzyme is Aci I, Bst UI, Hha I, HinP1, Hpa II, Hpy 99I, Ava I, Bce Al, Bsa HI, Bsi E1, Hga I, or a mixture thereof.

12. The method of claim 1, wherein the optional additional nuclease comprises a restriction endonuclease.

13. The method of claim 1, wherein the optional additional nuclease comprises McrBc.

14. The method of claim 1, wherein the optional additional nuclease comprises DNAase I.

15. The method of claim 1, wherein the incorporating step (c)(1) is further defined as:

generating single stranded nucleic acid molecules from at least some of the digested DNA molecules by heating;

subjecting the single stranded nucleic acid molecules to a plurality of primers to form a single stranded nucleic acid molecule/primer mixture, wherein the primers comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality and wherein the primers comprise a constant nucleic acid sequence and a variable nucleic acid sequence; and

subjecting said single stranded nucleic acid molecule/primer mixture to a polymerase to generate a plurality of molecules comprising the constant nucleic acid sequence at each end.

16. The method of claim 1, wherein the incorporating step (c)(2) is further defined as providing in a single incubation the following:

at least one digested DNA molecule;

an oligonucleotide comprising an inverted repeat and a loop;

DNA polymerase comprising 3′

-5′

exonuclease activity;

DNA ligase;

dNTPs;

ATP; and

a buffer suitable for activity of the polymerase and ligase.

17. The method of claim 16, wherein the incubation further comprises uracil-DNA-glycosylase and wherein said buffer is further suitable for activity of the glycosylase.

18. The method of claim 16, wherein said incubation further comprises a mixture of methylation-sensitive restriction enzymes and wherein said buffer is further suitable for activity of the restriction enzymes.

19. The method of claim 16, wherein the incubation further comprises uracil-DNA-glycosylase and a mixture of methylation-sensitive restriction enzymes and wherein said buffer is further suitable for activity of the glycosylase and the restriction enzymes.

20. The method of claim 1, wherein the inverted repeat comprises at least one replication stop.

21. The method of claim 20, wherein the replication stop is generated in the synthesis of the oligonucleotide.

22. The method of claim 21, wherein the synthesis comprises incorporation of a non-replicable region.

23. The method of claim 20, wherein the replication stop is generated by converting deoxyuridine to an abasic site.

24. The method of claim 23, wherein the converting step employs uracil-DNA-glycosylase.

25. The method of claim 1, wherein the amplifying step comprises polymerase chain reaction.

26. The method of claim 1, further comprising the step of heating the digested DNA molecules and/or the first modified DNA molecules, wherein the extension has not occurred in said oligonucleotide-linked molecules, to a temperature that causes denaturation of a specific fraction of the DNA.

27. The method of claim 4, wherein the analyzing step comprises sequencing, quantitative real-time polymerase chain reaction, ligation chain reaction, ligation-mediated polymerase chain reaction, probe hybridization, probe amplification, microarray hybridization, restriction enzyme digestion, or a combination thereof.

28. The method of claim 1, further comprising:

digesting the amplified first modified DNA molecules with the at least one methylation-sensitive restriction enzyme to produce amplified first modified fragments;

incorporating a nucleic acid molecule onto ends of the amplified first modified fragments to provide second modified DNA molecules, by one of the following;

(1) incorporating an adaptor having a nonblocked 3′

end to produce adaptor-linked fragments, wherein the 5′

end of the fragment is attached to the nonblocked 3′

end of the adaptor, leaving a nick site between the juxtaposed 3′

end of the fragment and a 5′

end of the adaptor, and polymerizing the 3′

end of the molecule from the nick site;

or

(2) incorporating an oligonucleotide comprising an inverted repeat and a loop, under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the fragment, thereby producing an oligonucleotide-linked fragment comprising a nick having a 3′

hydroxyl group, wherein there is polymerization from the 3′

hydroxyl group of at least part of the oligonucleotide-linked fragment; and

amplifying the second modified DNA molecules to provide amplified second modified DNA molecules.

29. The method of claim 28, further comprising analyzing the amplified second modified DNA molecules to determine the methylation status of the provided DNA molecule.

30. The method of claim 28, further comprising the step of heating the amplified first modified DNA molecules and/or the second modified DNA molecules, wherein the polymerization in the adaptor-linked fragment or the oligonucleotide-linked fragment has not occurred, to a temperature that causes denaturation of a specific fraction of the DNA.

31. The method of claim 28, wherein the incorporating step (2) is further defined as providing in a single incubation the following:

at least one amplified first modified fragment;

an oligonucleotide comprising an inverted repeat and a loop;

DNA polymerase comprising 3′

-5′

exonuclease activity;

DNA ligase;

dNTPs;

ATP; and

a buffer suitable for activity of the polymerase and ligase.

32. The method of claim 31, wherein the incubation further comprises uracil-DNA-glycosylase and wherein said buffer is further suitable for activity of the glycosylase.

33. The method of claim 31, wherein said incubation further comprises a mixture of methylation-sensitive restriction enzymes and wherein said buffer is further suitable for activity of the restriction enzymes.

34. The method of claim 31, wherein the incubation further comprises uracil-DNA-glycosylase and a mixture of methylation-sensitive restriction enzymes and wherein said buffer is further suitable for activity of the glycosylase and the restriction enzymes.

35. The method of claim 1, further defined as determining the methylation status of at least part of the provided DNA molecule.

36. The method of claim 1, further defmed as performing the method with a provided molecule from a sample of an individual with a medical condition in comparison to performing the method with a control.

37. The method of claim 36, wherein the medical condition is cancer.

38. The method of claim 1, wherein the provided DNA molecule comprises a promoter, a CpG island, or both.

39. The method of claim 1, wherein the provided DNA is further defined as bisulfite-converted DNA.

40. A method of preparing a DNA molecule, comprising:

(a) providing a DNA molecule;

(b) digesting the molecule with one or more methylation-specific restriction enzymes to provide DNA fragments;

(c) incorporating a nucleic acid molecule into the DNA fragments to provide first modified DNA molecules, by a method comprising;

(1) incorporating at least one primer from a plurality of primers, said primer comprising a 5′

constant sequence and a 3′

variable sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality;

(2) incorporating a first adaptor having a nonblocked 3′

end to produce first adaptor-linked molecules, wherein the 5′

end of the fragment is attached to the nonblocked 3′

end of the adaptor, leaving a nick site between the juxtaposed 3′

end of the fragment and a 5′

end of the first adaptor, and polymerizing the 3′

end of the fragment from the nick site;

or

(3) incorporating an oligonucleotide comprising an inverted repeat and a loop, under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the fragment, thereby producing an oligonucleotide-linked molecule comprising a nick having a 3′

hydroxyl group, wherein there is polymerization from the 3′

hydroxyl group of at least part of the oligonucleotide-linked molecule; and

(d) amplifying at least one first modified DNA molecule to provide amplified DNA molecules.

41. The method of claim 40, wherein the amplifying step utilizes a primer that is complementary to the incorporated nucleic acid molecule.

42. The method of claim 40, wherein the method further comprises the step of analyzing at least one of the amplified DNA molecules to determine the methylation status of the provided DNA.

43. The method of claim 40, wherein the methylation-specific endonuclease is McrBc.

44. A method of preparing a DNA molecule, comprising:

(a) providing one or more nucleic acid molecules;

(b) incorporating a nucleic acid molecule into the molecules by one or more of the following, wherein the incorporated molecule is resistant to bisulfite conversion, to provide first modified DNA molecules;

(1) incorporating sequence by attaching a first adaptor having a nonblocked 3′

end to the ends of the molecule to produce first adaptor-linked molecules, wherein the 5′

end of the molecule is attached to the nonblocked 3′

end of the adaptor, leaving a nick site between the juxtaposed 3′

end of the molecule and a 5′

end of the first adaptor, and extending the 3′

end of the molecule from the nick site;

or

(2) incorporating sequence by providing an oligonucleotide comprising an inverted repeat and a loop, under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the DNA molecule, thereby producing an oligonucleotide-linked DNA molecule comprising a nick having a 3′

hydroxyl group, wherein there is polymerization from the 3′

hydroxyl group of at least part of the oligonucleotide-linked DNA molecule;

(c) providing sodium bisulfite to said first modified nucleic acid molecules, wherein the unmethylated cytosines in said nucleic acid molecules are converted to uracil, thereby producing bisulfite-converted single-stranded nucleic acid molecules; and

(d) amplifying one or more of the bisulfite-converted molecules.

45. The method of claim 44, further comprising the step of analyzing the amplified bisulfite-converted molecules to determine the methylation status of the provided nucleic acid molecule.

46. The method of claim 44, further defined as performing the method with a provided molecule from a cancer sample, or with a provided molecule from a sample suspected of being a cancer sample, in comparison to performing the method with a control.

47. The method of claim 44, further comprising digesting the nucleic acid molecules, the first modified DNA molecules, or the bisulfite-converted molecules with a methylation-sensitive restriction enzyme.

48. The method of claim 44, further comprising analyzing the digested nucleic acid molecules, the digested first modified DNA molecules, or the digested bisulfite-converted molecules to determine the methylation status of the provided nucleic acid molecules.

49. A method of preparing a DNA molecule, comprising the steps of:

(1) providing a DNA molecule;

(2) altering the molecule in a single incubation to produce oligonucleotide-linked molecules, said incubation comprising two of more of the following;

(a) modifying the ends of the DNA molecules to provide attachable ends;

(b) repairing nicks and/or gaps within the DNA molecules;

(c) attaching a first oligonucleotide comprising a known sequence and a nonblocked 3′

end to the ends of the DNA molecules to produce oligonucleotide-linked molecules, wherein the 5′

end of the DNA is attached to the nonblocked 3′

end of the oligonucleotide, leaving a nick site between the juxtaposed 3′

end of the DNA molecule and a 5′

end of the oligonucleotide; and

(d) polymerizing from the 3′

end of the molecule from the nick site;

(3) digesting the oligonucleotide-linked DNA molecules with a mixture of methylation-sensitive restriction enzymes that do not cleave within the attached first oligonucleotide; and

(4) amplifying the digested first oligonucleotide-linked DNA molecules with a primer complementary to at least a portion of the stem region of the oligonucleotide to produce amplified oligonucleotide-linked fragments.

50. The method of claim 49, further comprising the step of analyzing at least part of the sequence of the amplified oligonucleotide-linked fragments.

51. The method of claim 49, wherein the DNA molecule is provided from body fluid or tissue.

52. The method of claim 51, wherein the body fluid comprises blood, serum, urine, cerebrospinal fluid, nipple aspirate, sweat, or saliva.

53. The method of claim 51, wherein the tissue comprises biopsy, surgical sample, or cheek scrapings.

54. The method of claim 49, wherein digestion of the DNA molecules with the mixture of methylation-sensitive restriction enzymes occurs during the altering step.

55. The method of claim 49, wherein the attached oligonucleotide comprises a non-replicable region in its loop.

56. The method of claim 55, wherein the non-replicable region is generated during the altering of the DNA molecule.

57. The method of claim 55, wherein the non-replicable region comprises at least one abasic site.

58. The method of claim 57, wherein the abasic site is generated from deoxyuridines comprised within the 5′

stem and loop region of the oligonucleotide.

59. The method of claim 49, wherein the 3′

end of the DNA molecule is extended from the nick site up to a non-replicable region of the oligonucleotide.

60. The method of claim 49, wherein said amplifying comprises a first heating step to fragment abasic regions of the oligonucleotide-linked molecules.

61. The method of claim 49, further comprising a step wherein sodium bisulfite is provided to said oligonucleotide-linked molecules, wherein the unmethylated cytosines in said oligonucleotide-linked molecules are converted to uracil, thereby producing bisulfite-converted molecules.

62. The method of claim 49, wherein the oligonucleotide is further defined as comprising a 3′

stem region, wherein said 3′

stem region does not comprise guanine and wherein all cytosines are methylated.

63. The method of claim 49, further comprising a step of enriching for oligonucleotide-attached molecules comprising CpG-rich regions.

64. The method of claim 63, wherein said enrichment comprises heating.

65. The method of claim 64, wherein a subset of first oligonucleotide-attached molecules is denatured.

66. The method of claim 49, further comprising the step of comparing at least part of the sequence of the amplified oligonucleotide-linked fragments with a control DNA molecule that was not subjected to the digestion step.

67. The method of claim 49, further comprising:

digesting the amplified oligonucleotide-linked molecules with at least one of the methylation-sensitive restriction enzymes in the mixture;

attaching an adaptor to at least one digested oligonucleotide-linked molecule to produce an adaptor-linked fragment, wherein the 5′

end of the molecule is attached to the nonblocked 3′

end of the adaptor, leaving a nick site between the juxtaposed 3′

end of the fragment and a 5′

end of the adaptor;

polymerizing from the 3′

end of the molecule from the nick site; and

amplifying the adaptor-linked fragments with a primer complementary to at least part of the adaptor to produce amplified adaptor-linked fragments.

68. The method of claim 67, further comprising analyzing the amplified adaptor-linked fragments to determine the methylation status of the provided DNA.

69. The method of claim 67, wherein the adaptor comprises at least one end that is complementary to the ends of the digested amplified DNA fragments.

70. The method of claim 67, wherein the adaptor comprises at least one blunt end.

71. The method of claim 67, wherein the adaptor comprises one or known sequences.

72. The method of claim 71, wherein the one or more known sequences are substantially non-self complementary and substantially non-complementary to other second adaptors.

73. A method of preparing a DNA molecule, comprising:

(a) providing a DNA molecule resulting from apoptotic degradation;

(b) digesting the DNA molecule with at least one methylation-sensitive restriction enzyme and, optionally, an additional nuclease to provide digested DNA molecules;

(c) incorporating an adaptor having a nonblocked 3′

end to at least some of the digested DNA molecules to produce adaptor-linked molecules, wherein the 5′

end of the molecule is attached to the nonblocked 3′

end of the adaptor, leaving a nick site between the juxtaposed 3′

end of the molecule and a 5′

end of the adaptor, and polymerizing from the 3′

end of the molecule from the nick site, to produce a modified DNA molecule; and

(d) amplifying one or more of the modified DNA molecules to provide amplified modified DNA molecules.