Nucleic acid interaction analysis

• US 20070238101A1
• Filed: 03/13/2006
• Published: 10/11/2007
• Est. Priority Date: 03/13/2006
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Agency For Science Technology and Research
• Inventors: Ruan, Yijun, Ng, Huck, Fullwood, Melissa, Wei, Chia
• Granted Patent: US 8,071,296 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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, wherein the first and second tags are tags of a nucleic acid-protein complex.

2. The isolated oligonucleotide according to claim 1, wherein the first tag is of a first polynucleotide and the second tag is of a second polynucleotide, and wherein the first and second polynucleotides are polynucleotides of a nucleic acid-protein complex.

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

4. The isolated oligonucleotide according to claim 1, wherein the oligonucleotide further comprises at least one linker.

5. The isolated oligonucleotide according to claim 4, wherein the linker comprises at least one restriction enzyme recognition site.

6. The isolated oligonucleotide according to claim 4, wherein the at least one linker is inserted between the tags or positioned upstream to the first tag or downstream to the second tag.

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

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

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

terminus and a 3′

terminus of a first polynucleotide and the at least one second tag comprises a 5′

terminus and a 3′

terminus of a second polynucleotide, and wherein the first and second polynucleotides are polynucleotides of a nucleic acid-protein complex.

10. The isolated oligonucleotide according to claim 5, 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, the first and second polynucleotides being polynucleotides of a nucleic acid-protein complex.

11. The isolated oligonucleotide according to claim 5, 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′

terminus 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′

terminus of the second polynucleotide, the first and second polynucleotides being polynucleotides of a nucleic acid-protein complex.

12. The isolated oligonucleotide according to claim 5, 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′

terminus 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′

terminus of the second polynucleotide, the first and second polynucleotides being polynucleotides of a nucleic acid-protein complex.

13. The isolated oligonucleotide according to claim 5, 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′

terminus 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′

terminus of the second polynucleotide, the first polynucleotide further comprises a third recognition site recognised by a third restriction enzyme capable of cleaving the first polynucleotideto obtain a 5′

terminus of first polynucleotide, and the second polynucleotide comprises a fourth recognition site recognised by a fourth restriction enzyme capable of cleaving the second polynucleotideto obtain a 3′

terminus 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′

termini of the first polynucleotide and the at least one second tag obtained from ligating the 5′ and

3′

termini of the second polynucleotide.

14. The isolated oligonucleotide according to claim 1, wherein the nucleic acid-protein complex is part of a chromatin structure.

15. The isolated oligonucleotide according to claim 2, wherein the polynucleotides are DNA or RNA.

16. A vector comprising the oligonucleotide of claim 1.

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

17. 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, wherein the first and second tags are tags of a nucleic acid-protein complex.

18. The concatemer of oligonucleotides according to claim 17, wherein the first tag is a tag of a first polynucleotide and the second tag is a tag of a second polynucleotide, the first and second polynucleotides being from a nucleic acid-protein complex, and wherein each isolated oligonucleotide comprises at least one restriction enzyme recognition site.

19. The concatemer of oligonucleotides according to claim 18, wherein the at least one recognition site is included in at least one linker.

20. The concatemer of oligonucleotides according to claim 19, wherein the at least one linker is inserted between the tags or positioned upstream to the first tag and/or downstream to the second tag.

21. The concatemer of oligonucleotides according to claim 18, wherein the at least one restriction enzyme recognition site is for a type IIs restriction enzyme.

22. The concatemer of oligonucleotides according to claim 18, wherein the first tag comprises a 5′

terminus and a 3′

terminus from the first polynucleotide and the second tag further comprises a 5′

terminus and a 3′

terminus from the second polynucleotide.

24. A method of preparing at least one isolated oligonucleotide comprising:

(a) providing a nucleic acid-protein complex;

(b) preparing an oligonucleotide comprising 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; and

(c) isolating the oligonucleotide.

25. The method according to claim 24, wherein the first tag is obtained from a first polynucleotide and the second tag is obtained from a second polynucleotide, the first and second polynucleotides obtained from a nucleic acid-protein complex.

26. The method according to claim 25, wherein step (b) comprises:

(i) inserting at least one linker comprising at least one restriction enzyme recognition site, and

(ii) cleaving the first and second polynucleotides with a least one restriction enzyme recognizing the at least one recognition site in the linker to form an oligonucleotide comprising a first tag obtained from the first polynucleotide, a second tag obtained from the second polynucleotide and the linker.

27. The method according to claim 25, wherein step (b) comprises:

(i) inserting at least one linker comprising at least one restriction enzyme recognition site between the first polynucleotide and the second polynucleotide of the complex;

(ii) adding at least one restriction enzyme recognition site each to the 5′

terminus of the first polynucleotide and the 3′

terminus of the second polynucleotide;

(iii) cleaving the first and second polynucleotides with at least one restriction enzyme recognizing at least one recognition site to obtain cleaved fragments; and

(iv) ligating cleaved fragments to form the oligonucleotide comprising a first tag obtained from the first polynucleotide, a second tag obtained from the second polynucleotide, the tags comprising 5′ and

3′

termini of each polynucleotide, and the linker inserted between the tags.

28. The method according to claim 27, wherein the at least one restriction enzyme recognition site in step (ii) is part of an adaptor or of a vector.

29. The method according to claim 24, wherein the nucleic acid-protein complex is obtained by chromatin immunoprecipitation.

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

(a) providing a nucleic acid-protein complex;

(b) preparing an oligonucleotide comprising at least one first tag and at least one second tag, wherein the first tag is obtained from a first polynucleotide and the second tag obtained from a second polynucleotide, the first and second polynucleotides obtained from a nucleic acid-protein complex;

(c) sequencing the oligonucleotide; and

(d) 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.

31. The method according to claim 30, wherein step (b) comprises:

(i) inserting at least one linker comprising at least one restriction recognition site, and

(ii) cleaving the first and second polynucleotides with a least one restriction enzyme recognizing the at least one recognition site in the linker to form the oligonucleotide comprising a first tag obtained from the first polynucleotide, a second tag obtained from the second polynucleotide and the linker between the tags.

32. The method according to claim 30, wherein step (b) comprises:

(i) inserting at least one linker comprising at least one restriction enzyme recognition site between the first polynucleotide and the second polynucleotide of the complex;

(ii) adding at least one restriction enzyme recognition site each to the 5′

terminus of the first polynucleotide and the 3′

terminus of the second polynucleotide;

(iii) cleaving the first and second polynucleotides with at least one restriction enzyme recognizing at least one recognition site to obtain cleaved fragments; and

(iv) ligating cleaved fragments to form the oligonucleotide comprising a first tag obtained from the first polynucleotide, a second tag obtained from the second polynucleotide, the tags comprising 5′ and

3′

termini of each polynucleotide, and the linker inserted between the tags.

33. The method according to claim 32, wherein the at least one restriction enzyme recognition site in step (ii) is part of an adaptor or of a vector.

34. The method according to claim 30, wherein the nucleic acid-protein complex is obtained by chromatin immunoprecipitation.

35. The method according to claim 30, wherein the oligonucleotide is concatenated with at least one further oligonucleotide obtained by steps (a) to (b) before being sequenced.

36. The method according to claim 30, wherein the polynucleotides are located on the same chromosome or the polynucleotides are located on different chromosomes.