Methods for preparing amplifiable DNA molecules

• US 8,778,610 B2
• Filed: 02/27/2013
• Issued: 07/15/2014
• 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
• Inventors: Makarov, Vladimir L., Kamberov, Emmanuel, Tarrier, Brendan J.
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US13/779,459
• Publication Number:
  US 20130316913A1
• Time in Patent Office: 503 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 435/6.12
• 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 preparing 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 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;

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;

ligating an adaptor onto the end of at least some of the renatured GC-rich molecules to produce adaptor-ligated molecules, wherein the adaptor is further defined as a stem-loop oligonucleotide comprising an inverted repeat and a loop.

2. 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, or about, 50%.

3. The method of claim 1, wherein the first temperature is greater than, or about, 60°

C.

4. The method of claim 1, wherein the second temperature is lower than, or about, 85°

C.

5. The method of claim 1, wherein the ends of the renatured GC-rich molecules are polished prior to said ligating.

6. The method of claim 1, wherein the ligating is further defined as blunt-end ligating.

7. The method of claim 1, wherein the ligating is further defined as ligating with both strands of the DNA molecules and the adaptors.

8. The method of claim 1, 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.

9. The method of claim 1, 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.

10. The method of claim 1, further comprising subjecting the adaptor-ligated DNA molecules to one or more methylation-sensitive restriction enzymes.

11. The method of claim 1, further comprising subjecting the adaptor-ligated DNA molecules to one or more methylation-specific restriction enzymes.

12. The method of claim 1, wherein the adaptor is further defined as comprising a restriction endonuclease site.

13. The method of claim 12, wherein the endonuclease site is present in the inverted repeat.

14. The method of claim 12, further comprising subjecting the adaptor-ligated molecules to the restriction endonuclease.

15. The method of claim 1, wherein the plurality of DNA molecules that are provided are from a body fluid or tissue.

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

17. The method of claim 1, further comprising determining at least part of the sequence of one or more of the adaptor-ligated molecules.

18. The method of claim 17, wherein the determined sequence comprises a regulatory sequence.

19. The method of claim 17, wherein the determining step provides diagnostic information for an individual.

20. The method of claim 19, wherein the diagnostic information comprises cancer diagnosis information for the individual.

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

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

23. The method of claim 1, further comprising subjecting the adaptor-ligated molecules to an amplification reaction to provide amplified molecules.

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