Isolation of CpG islands by thermal segregation and enzymatic selection-amplification method

• US 20070031858A1
• Filed: 03/02/2006
• Published: 02/08/2007
• Est. Priority Date: 08/02/2005
• 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: Makarov, Vladimir, Kamberov, Emmanuel, Tarrier, Brendan
• Granted Patent: US 8,409,804 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases

Abstract

The present invention concerns isolation, library preparation and selective amplification from a compositionally heterogeneous pool of DNA fragments of a fraction of molecules, such as those originating from promoter CpG islands and characterized by a high GC content. In particular, the process utilizes a heat-induced segregation of DNA molecules into GC-poor, single-stranded molecule fractions and GC-rich, double-stranded molecule fractions, with subsequent enzymatic conversion of the GC-rich, double-stranded DNA molecules into a library, and, optionally, amplification. In specific embodiments, the isolation process is used to generate promoter-enriched genomic and methylome libraries for research and diagnostic applications, for example.

Claims

1. A method of amplifying a plurality of amplifiable DNA molecules, comprising:

providing a plurality of DNA molecules, said plurality having molecules comprising one or more regions that are GC-poor and having molecules comprising one or more regions that are GC-rich;

subjecting the plurality of DNA molecules to sufficient conditions to denature GC-poor regions but not to denature GC-rich regions, thereby producing GC-rich regions suitable for amplification; and

subjecting the plurality to amplification conditions such that the denatured GC-poor regions are substantially not amplified and such that one or more of the non-denatured or partially denatured GC-rich regions are amplified.

2. The method of claim 1, wherein the conditions to denature GC-poor regions but not to denature GC-rich regions comprise temperature sufficient to denature GC-poor regions but not to denature GC-rich regions, pressure sufficient to denature GC-poor regions but not to denature GC-rich regions, pH sufficient to denature GC-poor regions but not to denature GC-rich regions, or a combination thereof.

3. The method of claim 1, wherein the conditions to denature GC-poor regions but not to denature GC-rich regions comprise temperature sufficient to denature GC-poor regions but not to denature GC-rich regions.

4. The method of claim 1, wherein the subjecting the plurality of DNA molecules to sufficient conditions to denature GC-poor regions but not to denature GC-rich regions is further defined as:

subjecting the plurality to a first temperature such that the GC- poor regions are substantially denatured and such that the GC-rich regions are undenatured or are denatured only in part; and

subjecting the plurality to a second temperature such that at least part of the GC-poor regions incompletely renature and such that at least part of the GC-rich regions substantially completely renature, thereby producing renatured amplifiable GC-rich molecules.

5. The method of claim 1, wherein the molecules comprising GC-rich regions are further defined as comprising one or more regions having GC content greater than about 50%.

6. The method of claim 4, wherein the first temperature is greater than about 60°

C.

7. The method of claim 4, wherein the second temperature is lower than about 85°

C.

8. The method of claim 4, further comprising ligating an adaptor onto the end of at least some of the renatured GC-rich molecules to produce adaptor-ligated molecules.

9. The method of claim 8, wherein the ends of the renatured GC-rich molecules are polished prior to said ligating.

10. The method of claim 8, wherein the ligating is further defined as blunt-end ligating.

11. The method of claim 8, wherein the renatured GC-rich molecules are further defined as restriction enzyme fragments, and wherein the adaptors are suitable for ligation onto the respective digested fragment ends.

12. The method of claim 8, wherein the ligating is further defined as ligating with both strands of the DNA molecules and the adaptors.

13. The method of claim 8, wherein the ligating is further defined as ligating with only one strand of each molecule, said one strand being the 5′

end of the DNA molecules and the 3′

end of the adaptors, wherein the method further comprises 3′

extension of a nick in the adaptor-ligated molecules.

14. The method of claim 4, wherein the GC-poor regions that are substantially denatured are further defined as having one or more regions that are single stranded following said subjecting step, and wherein the single stranded regions are subjected to a single strand-specific endonuclease.

15. The method of claim 8, further comprising subjecting the adaptor-ligated DNA molecules to one or more methylation-sensitive restriction enzymes.

16. The method of claim 8, further comprising subjecting the adaptor-ligated DNA molecules to one or more methylation-specific restriction enzymes.

17. The method of claim 8, wherein the adaptor is further defined as a stem-loop oligonucleotide comprising an inverted repeat and a loop.

18. The method of claim 8, wherein the adaptor is further defined as comprising a restriction endonuclease site.

19. The method of claim 18, wherein the endonuclease site is present in the inverted repeat.

20. The method of claim 18, further comprising subjecting the adaptor-ligated molecules to the restriction endonuclease.

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

22. The method of claim 1, wherein the plurality of DNA molecules comprise known sequences at the ends of the molecules.

23. The method of claim 1, further comprising determining at least part of the sequence of one or more of the amplified molecules.

24. The method of claim 23, wherein the determined sequence comprises a regulatory sequence.

25. The method of claim 23, wherein the determining step provides diagnostic information for an individual.

26. The method of claim 25, wherein the diagnostic information comprises cancer diagnosis information for the individual.

27. The method of claim 1, wherein the GC-rich region comprises at least part of regulatory sequence.

28. The method of claim 1, wherein the GC-rich region comprises at least part of a CpG island.

29. A method of amplifying a CpG island from a molecule, comprising:

providing a plurality of DNA molecules, said plurality comprising at least one molecule having at least one CpG island and said plurality having at least one molecule comprising one or more regions that are GC-poor;

subjecting the plurality of DNA molecules to sufficient conditions to denature GC-poor regions but not to substantially denature the CpG island, thereby rendering the CpG island suitable for amplification; and

subjecting the plurality to amplification conditions such that the denatured GC-poor regions are substantially not amplified and such that the non-denatured or partially denatured CpG-island is amplified.

30. The method of claim 29, wherein the CpG island is further defined comprising at least part of a regulatory sequence.

31. A method of preparing a library, comprising:

providing a plurality of DNA molecules, said plurality having molecules comprising one or more regions that are GC-poor and having molecules comprising one or more regions that are GC-rich;

subjecting the plurality of DNA molecules to sufficient conditions to denature GC-poor regions but not to denature GC-rich regions, thereby producing GC-rich regions suitable for amplification; and

subjecting the plurality to amplification conditions such that the denatured GC-poor regions are substantially not amplified and such that one or more of the non-denatured or partially denatured GC-rich regions are amplified.

32. The method of claim 31, further comprising ligating an adaptor onto the end of at least some of the renatured GC-rich molecules to produce adaptor-ligated molecules.

33. The method of claim 32, wherein the ends of the renatured GC-rich molecules are polished prior to said ligating.

34. The method of claim 32, wherein the ligating is further defined as blunt-end ligating.

35. The method of claim 32, wherein the renatured GC-rich molecules are further defined as restriction enzyme fragments, and wherein the adaptors are suitable for ligation onto the respective digested fragment ends.