Nucleic acid interaction analysis

• US 8,071,296 B2
• Filed: 03/13/2006
• Issued: 12/06/2011
• Est. Priority Date: 03/13/2006
• Status: Expired due to Fees

• Current Assignee: Agency For Science Technology and Research (Government of Singapore)
• Original Assignee: Agency For Science Technology and Research (Government of Singapore)
• Inventors: Ruan, Yijun, Fullwood, Melissa Jane, Wei, Chia Lin
• Primary Examiner(s): Benzion, Gary
• Assistant Examiner(s): WILDER, CYNTHIA B
• Application Number:
  US11/373,519
• Publication Number:
  US 20070238101A1
• Time in Patent Office: 2,094 Days
• Field of Search: 536/23.1
• US Class Current: 435/6.1
• 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: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2531/113 PCR

Abstract

The present invention provides an isolated oligonucleotide and a method using the isolated oligonucleotide to detect and/or identify at least two polynucleotides from a nucleic acid-protein complex. The oligonucleotide comprises at least one first tag and at least one second tag, wherein the first and second tags are obtained from a nucleic acid-protein complex.

Claims

1. An isolated oligonucleotide comprising at least one first tag and at least one second tag, and at least one linker inserted between the first and second tags, wherein the first and second tags comprise an identifiable sequence of 8-20 base pairs, wherein the first tag comprises at least one most terminal nucleotide sequence from a first polynucleotide and the second tag comprises at least one most terminal nucleotide sequence from a second polynucleotide, wherein the first and second polynucleotides each are capable of binding with at least one protein of a nucleic acid-protein complex, wherein the nucleic acid-protein complex is part of a chromatin structure or is obtained through chromatin immunoprecipitation or both, and wherein the first and second polynucleotides comprise distal interacting regions from the same or different chromosomes.

2. The isolated oligonucleotide according to claim 1, wherein the oligonucleotide further comprises at least one restriction enzyme recognition site.

3. The isolated oligonucleotide according to claim 2, wherein the linker comprises the at least one restriction enzyme recognition site.

4. The isolated oligonucleotide according to claim 3, wherein the at least one restriction enzyme recognition site is for a type IIs restriction enzyme.

5. The isolated oligonucleotide according to claim 3, wherein the at least one restriction enzyme recognition site is for a homing restriction enzyme.

6. The isolated oligonucleotide according to claim 1, wherein the at least one first tag comprises a 5′

most terminal nucleotide sequence and a 3′

most terminal nucleotide sequence of the first polynucleotide and the at least one second tag comprises a 5′

most terminal nucleotide sequence and a 3′

most terminal nucleotide sequence of the second polynucleotide.

7. The isolated oligonucleotide according to claim 3, wherein the linker comprises a first restriction recognition site recognized by a restriction enzyme capable of cleaving a first polynucleotide to obtain the first tag, and a second restriction recognition site recognized by a restriction enzyme capable of cleaving a second polynucleotide to obtain the second tag.

8. The isolated oligonucleotide according to claim 3, wherein the linker comprises a first restriction recognition site recognized by a first restriction enzyme capable of cleaving a first polynucleotide to obtain a 3′

most terminal nucleotide sequence of the first polynucleotide, and a second restriction recognition site recognized by a second restriction enzyme capable of cleaving a second polynucleotide to obtain a 5′

most terminal nucleotide sequence of the second polynucleotide.

9. The isolated oligonucleotide according to claim 3, wherein the linker comprises a first restriction recognition site recognized by a first restriction enzyme capable of cleaving a first polynucleotide to obtain a 5′

most terminal nucleotide sequence of the first polynucleotide, and a second restriction recognition site recognized by a second restriction enzyme capable of cleaving a second polynucleotide to obtain a 3′

most terminal sequence of the second polynucleotide.

10. The isolated oligonucleotide according to claim 3, wherein the linker comprises a first restriction recognition site recognized by a first restriction enzyme capable of cleaving a first polynucleotide to obtain a 3′

most terminal nucleotide sequence of the first polynucleotide, and a second restriction recognition site recognized by a second restriction enzyme capable of cleaving a second polynucleotide to obtain a 5′

most terminal nucleotide sequence of the second polynucleotide, the first polynucleotide further comprises a third recognition site recognized by a third restriction enzyme capable of cleaving the first polynucleotide to obtain a 5′

most terminal nucleotide sequence of the first polynucleotide, and the second polynucleotide comprises a fourth recognition site recognized by a fourth restriction enzyme capable of cleaving the second polynucleotide to obtain a 3′

terminus most terminal nucleotide sequence of the second polynucleotide;

the first and second polynucleotides being polynucleotides of a nucleic acid-protein complex;

the at least one first tag obtained from ligating the 5′ and

3′

most terminal nucleotide sequences of the first polynucleotide and the at least one second tag obtained from ligating the 5′ and

3′

most terminal nucleotide sequences of the second polynucleotide.

11. A vector comprising the oligonucleotide of claim 1.

17. An oligonucleotide library comprising at least one oligonucleotide according to claim 1.

18. A method of detecting and/or identifying at least two polynucleotides of a nucleic acid-protein complex, the method comprising:

(a) providing the isolated oligonucleotide of claim 1;

(b) sequencing the oligonucleotide; and

(c) mapping the at least two polynucleotides based on the nucleotide sequences of the first and second tags,

thereby detecting and/or identifying the at least two polynucleotides.

19. The method according to claim 18, wherein the nucleic acid-protein complex is obtained by chromatin immunoprecipitation.

20. The method according to claim 18, wherein the oligonucleotide is concatenated with at least one further oligonucleotide of step (a) before being sequenced.

21. The method according to claim 18, wherein the polynucleotides are located on the same chromosome or the polynucleotides are located on different chromosomes.

22. The isolated oligonucleotide of claim 1, wherein the first and second tags comprise an identifiable sequence of 8-16 base pairs.

23. The isolated oligonucleotide of claim 1, wherein the first and second tags comprise an identifiable sequence of 16-20 base pairs.

12. A concatemer of oligonucleotides comprising at least two isolated oligonucleotides, each isolated oligonucleotide comprising at least one first tag and at least one second tag, and at least one linker inserted between the first and second tags, wherein the first and second tags comprise an identifiable sequence of 8-20 base pairs, wherein the first tag comprises at least one most terminal nucleotide sequence from a first polynucleotide and the second tag comprises at least one most terminal nucleotide sequence from a second polynucleotide, and wherein the first and the second polynucleotides each are capable of binding with at least one protein of a nucleic acid-protein complex which is part of a chromatin structure or obtained through chromatin immunoprecipitation or both, and wherein the first and second polynucleotides comprise distal interacting regions from the same or different chromosomes.

13. The concatemer of oligonucleotides according to claim 12, wherein each isolated oligonucleotide comprises at least one restriction enzyme recognition site.

14. The concatemer of oligonucleotides according to claim 13, wherein for each oligonucleotide, the at least one recognition site is included in the linker.

15. The concatemer of oligonucleotides according to claim 13, wherein for each oligonucleotide, the at least one restriction enzyme recognition site is for a type IIs restriction enzyme.

16. The concatemer of oligonucleotides according to claim 12, wherein for each oligonucleotide the first tag comprises a 5′

most terminal nucleotide sequence and a 3′

most terminal nucleotide sequence from the first polynucleotide and the second tag further comprises a 5′

most terminal nucleotide sequence and a 3′

most terminal nucleotide sequence from the second polynucleotide.

24. The concatemer of oligonucleotides of claim 12, wherein the first and second tags comprise an identifiable sequence of 8-16 base pairs.

25. The concatemer of oligonucleotides of claim 12, wherein the first and second tags comprise an identifiable sequence of 16-20 base pairs.