Nucleic acid interaction analysis

• US 8,263,367 B2
• Filed: 01/25/2008
• Issued: 09/11/2012
• Est. Priority Date: 01/25/2008
• Status: Expired due to Fees

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Agency For Science Technology and Research
• Inventors: Ruan, Yijun, Wei, Chialin, Fullwood, Melissa Jane, Liu, Jun
• Primary Examiner(s): MUMMERT, STEPHANIE KANE
• Application Number:
  US12/020,071
• Publication Number:
  US 20090191598A1
• Time in Patent Office: 1,691 Days
• Field of Search: None
• US Class Current: 435/91.52
• 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 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

The present invention provides at least one isolated linear composite nucleic acid molecule comprising at least one first tag from at least one first nucleic acid molecule and at least one second tag from at least one second nucleic acid molecule, wherein the first and second nucleic acids interact in a nucleic acid mixture; and wherein the first and second tags are from different nucleic acid molecules. The invention also provides a method of producing at least one isolated linear composite nucleic acid and to a method of detecting and/or identifying nucleic acid interactions.

Claims

1. A method of preparing at least one isolated linear composite nucleic acid molecule comprising a first tag from a first nucleic acid molecule and a second tag from a second nucleic acid molecule, wherein the first and second nucleic acid molecules interact in a nucleic acid-protein complex and wherein the first and second tags are from different nucleic acid molecules;

comprising the steps of;

(i) providing at least one nucleic acid-protein complex comprising at least one first nucleic acid molecule interacting with at least one second nucleic acid molecule;

(ii) (a) ligating at least one linker with at least one non-ligatable end to at least one end of the nucleic acid molecule(s) in the nucleic acid mixture;

(b) converting the non-ligatable end(s) of the ligated linker(s) to make them ligatable; and

(c) ligating the linker(s) to ligate the end(s) of the nucleic acid molecules to form at least one linker-ligated linear composite nucleic acid molecule comprising at least one linker between the two nucleic acid molecules;

(iii) size selecting for linker-ligated linear composite nucleic and molecules comprising a first tag from a first nucleic acid molecule; and

a second tag from a second nucleic acid molecule; and

(iv) ligating at least one adapter to at least one end of the linker-ligated linear composite nucleic acid molecule(s) and isolating at least one linear composite nucleic acid molecule with both adapter(s) and linker(s) ligated, wherein the first and second tags of the linear composite nucleic acid molecule(s) are from different nucleic and molecules.

2. The method according to claim 1, wherein isolating the linear composite nucleic acid molecule(s) comprises using a binding system comprising at least two components, wherein at least one first component is coupled to the adapter, and at least one second component binds the first component to isolate the linear composite nucleic acid molecule(s).

3. The method according to claim 1, wherein isolating the linear nucleic acid molecule(s) comprises using a binding system comprising at least two components, wherein at least one first component is coupled to the linker, and at least one second component binds the first component to isolate the linear composite nucleic acid molecule(s).

4. The method according to claim 2, wherein the binding system comprises the components of streptavadin-biotin, avidin-biotin, protein-antibody and/or magnet-magnetic material.

5. The method according to claim 3, wherein the binding system comprises the components of streptavadin-biotin, avidin-biotin, protein-antibody and/or magnet-magnetic material.

6. The method according to claim 1, wherein step (ii) comprises:

(a) ligating at least one dephosphorylated linker to at least one end of the nucleic acid molecules in the nucleic acid mixture;

(b) phosphorylating the dephosphorylated end of the ligated linker(s); and

(c) ligating the phosphorylated linker(s) to ligate the ends of the nucleic acid molecules to form at least one linker-ligated linear composite nucleic acid molecule comprising at least one linker between the ligated nucleic acid molecules.

7. The method according to claim 1, wherein step (ii) comprises:

(a) ligating at least one dephosphorylated linker comprising at least one restriction enzyme site to at least one end of the nucleic acid molecules in the nucleic acid mixture;

(b) cutting the ligated linker(s) with the restriction enzyme(s) for the site(s) to produce cohesive ends; and

(c) ligating the cohesive ends of the linker(s) to ligate the ends of the nucleic acid molecules to form at least one linker-ligated linear composite nucleic acid molecule comprising at least one linker between the ligated nucleic acid molecules.

8. The method according to claim 1, wherein step (iii) comprises selecting for linear composite nucleic acid molecules of 200-500 bp.

9. The method according to claim 1, further comprising the step of amplifying the linear composite nucleic acid molecules.

10. The method according to claim 1, further comprising sequencing at least one linear composite nucleic acid molecules.

11. The method according to claim 10, wherein sequencing the linear composite nucleic acid molecules comprises sequencing from different regions of the linear composite nucleic acid molecules to obtain sequence information for the two tags.

12. The method according to claim 11, further comprising mapping the sequenced tags of the linear composite nucleic acid molecule to at least one reference sequence.

13. The method according to claim 12, wherein the reference sequence includes at least one sequence database.

14. The method according to claim 13, wherein the sequence database is at least one genome sequence database.

15. The method according to claim 1, wherein the nucleic acid-protein complex comprises chromatin.

16. The method according to claim 1, wherein the nucleic acid-protein complex is obtained by chromatin immunoprecipitation.