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

• US 9,708,652 B2
• Filed: 04/09/2013
• Issued: 07/18/2017
• Est. Priority Date: 03/08/2004
• Status: Expired due to Fees

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Rubicon Genomics, Inc (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Sun, Tong, Pinter, Jonathan H., Tarrier, Brendan J., Bruening, Eric E., Kurihara, Takao, Tesmer, Tim, M'Mwirichia, Joseph, Makarov, Vladimir L., Kamberov, Emmanuel
• Primary Examiner(s): Priest, Aaron
• Application Number:
  US13/859,034
• Publication Number:
  US 20130309668A1
• Time in Patent Office: 1,561 Days
• Field of Search: None
• 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 particular, the method employs preparation of a DNA molecule by digesting the DNA molecule with at least one methylation-sensitive restriction enzyme; incorporating a nucleic acid molecule into at least some of the digested DNA molecules by either (1) incorporating at least one primer from a plurality of primers that have a 5′ constant sequence and a 3′ variable sequence, wherein the primers are substantially non-self-complementary and substantially non-complementary to other primers in the plurality; or (2) incorporating an oligonucleotide having an inverted repeat and a loop under conditions wherein the oligonucleotide becomes blunt-end ligated to one strand of the digested DNA molecule, followed by polymerization from a 3′ hydroxyl group present in a nick in the oligonucleotide-linked molecule; and amplifying one or more of the DNA molecules.

Claims

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

(1) providing a DNA molecule;

(2) providing an oligonucleotide-linked DNA molecule in a single incubation, said single incubation comprising;

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

(b) ligating a first oligonucleotide comprising a known sequence and a nonblocked 3, end to an end of the DNA molecule to produce an oligonucleotide-linked molecule, wherein the 5′

end of the DNA molecule is attached to the nonblocked 3, end of the first oligonucleotide, leaving a nick site between a juxtaposed 3′

end of the DNA molecule and a 5′

end of the first oligonucleotide; and

(c) extending the juxtaposed 3, end of the DNA molecule from the nick site by polymerization;

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

(4) amplifying the digested oligonucleotide-linked molecule with a primer complementary to at least a portion of the known sequence of the first oligonucleotide to produce amplified oligonucleotide-linked molecules.

2. The method of claim 1, further comprising the step of determining at least part of the sequence of the amplified oligonucleotide-linked molecules.

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

4. The method of claim 3, wherein the body fluid comprises blood, serum, urine, cerebrospinal fluid, nipple aspirate, sweat, or saliva.

5. The method of claim 3, wherein the tissue comprises biopsy, surgical sample, or cheek scrapings.

6. The method of claim 1, wherein digestion of the oligonucleotide-linked molecule with the mixture of methylation-sensitive restriction enzymes occurs during step (2).

7. The method of claim 1, wherein the oligonucleotide-linked molecule comprises a non-replicable region in the first oligonucleotide portion.

8. The method of claim 7, wherein the non-replicable region is generated during step (2).

9. The method of claim 7, wherein the non-replicable region comprises at least one abasic site.

10. The method of claim 9, wherein the at least one abasic site is generated from at least one deoxyuridine located within the non-replicable region of the first oligonucleotide.

11. The method of claim 7, wherein the 3′

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

12. The method of claim 9, wherein the amplifying step comprises a first heating step to fragment the at least one abasic region of the oligonucleotide-linked molecule.

13. The method of claim 1, further comprising a step wherein sodium bisulfite is provided to said oligonucleotide-linked molecule, wherein any unmethylated cytosines in said oligonucleotide-linked molecule are converted to uracil, thereby producing a bisulfite-converted molecule.

14. The method of claim 1, wherein the first 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.

15. The method of claim 1, further comprising a step of enriching for oligonucleotide-linked molecules comprising CpG-rich regions.

16. The method of claim 1, further comprising a step of comparing at least part of a sequence of the amplified oligonucleotide-linked molecules with a control DNA molecule that was not subjected to the digesting step.

17. The method of claim 6, wherein the single incubation comprises the DNA molecule, a DNA polymerase, a DNA ligase, the first oligonucleotide, and the mixture of methylation-sensitive restriction enzymes.

18. The method of claim 1, wherein the first oligonucleotide is a stem-loop adaptor having an inverted repeat and a loop.

19. The method of claim 18, wherein the inverted repeat comprises a 5′

stem region and a 3′

stem region that is complementary to the 5′

stem region.

20. The method of claim 19, wherein the first oligonucleotide comprises a non-replicable base in the loop region.

21. The method of claim 20, wherein the first oligonucleotide further comprises a non-replicable base within the 5′

stem region.

22. The method of claim 1, wherein the first oligonucleotide does not contain a loop.

23. The method of claim 1, wherein the DNA molecule has been fragmented mechanically or chemically.

24. The method of claim 23, wherein the DNA molecule has been fragmented by sonication.

25. The method of claim 1, wherein the DNA molecule has been fragmented by enzymatic fragmentation.

26. The method of claim 1, wherein the enzymatic fragmentation is by at least one site-specific restriction endonuclease.

27. The method of claim 1, wherein the enzymatic fragmentation is by a random endonuclease.

28. The method of claim 1, wherein the DNA molecule has been fragmented in vivo.

29. The method of claim 28, wherein the DNA molecule is the product of apoptosis-induced degradation.

30. The method of claim 1, wherein the extending of step (2)(d) comprises nick translation polymerization.

31. The method of claim 1, wherein the extending of step (2)(d) comprises strand displacement polymerization.

32. The method of claim 1, wherein the incubation of step (2) further comprises repairing nicks and/or gaps within the DNA molecule.