Methods of identifying interactions between genomic loci

• US 9,434,985 B2
• Filed: 09/22/2009
• Issued: 09/06/2016
• Est. Priority Date: 09/25/2008
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, University Of Massachusetts, Whitehead Institute for Biomedical Research
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, University Of Massachusetts, Whitehead Institute for Biomedical Research
• Inventors: Van Berkum, Nynke, Dekker, Job, Lieberman, Erez, Gnirke, Andreas, Lander, Eric, Nusbaum, Chad, Williams, Louise, Melnikov, Alexandre, Giannoukos, Georgia
• Primary Examiner(s): Gussow, Anne
• Assistant Examiner(s): Brown, Mindy G
• Application Number:
  US13/121,158
• Publication Number:
  US 20130096009A1
• Time in Patent Office: 2,541 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence

Abstract

The disclosed Hi-C protocol can identify genomic loci that are spatially co-located in vivo. These spatial co-locations may include, but are not limited to, intrachromosomal interactions and/or interchromosomal interactions. Hi-C techniques may be applied to many different scales of interest. For example, on a large scale, Hi-C techniques can be used to identify long-range interactions between distant genomic loci.

Claims

1. A method for identifying interaction frequencies, comprising:

a) providing;

i) a nuclear matrix comprising a first genomic region and a second genomic region; and

ii) a junction marker labeled with an affinity marker for selective purification of a ligation product;

b) fragmenting said first genomic region and said second genomic region into a plurality of first genomic region fragments and a plurality of second genomic region fragments;

c) ligating said junction marker between at least one of said first genomic region fragments and at least one of said second genomic region fragments to create said ligation product;

d) purifying said ligation product with said affinity marker;

and

e) analyzing said ligation product under conditions such that a genomic interaction frequency for said ligation product is identified.

2. The method of claim 1, wherein said affinity marker comprises biotin.

3. The method of claim 1, wherein said first and second genomic regions are located on the same chromosome.

4. The method of claim 1, wherein said first and second genomic regions are located on different chromosomes.

5. The method of claim 1, wherein said interaction frequency identifies a long range interaction.

6. The method of claim 1, wherein said interaction frequency identifies a short range interaction.

7. The method of claim 1, wherein said interaction frequency identifies a close neighbor interaction.

8. The method of claim 1, wherein said nuclear matrix is derived from a human cell nucleus.

9. The method of claim 1, wherein said nuclear matrix is derived from a yeast cell nucleus.

10. The method of claim 1, further comprising contacting said junction marker with a ligand.

11. The method of claim 10, wherein said ligand comprises streptavidin.

12. The method of claim 1, further comprising digesting said nuclear matrix fragments with a restriction endonuclease wherein a first region fragment comprising a first sticky end and a second region fragment comprising a second sticky end are created.

13. The method of claim 12, further comprising filling in said first sticky end and said second sticky end to create a first blunt end and a second blunt end;

and ligating said first and second blunt ends, wherein said first region fragment and said second region fragment are joined.

14. The method of claim 1, further comprising digesting said nuclear matrix fragments with an exonuclease.

15. A method for identifying interaction frequencies, comprising:

a) crosslinking a nuclear matrix comprising a first genomic region and a second genomic region;

b) fragmenting said first genomic region and said second genomic region into a plurality of first genomic region fragments and a plurality of second genomic region fragments;

c) ligating a junction marker labeled with an affinity marker between at least one of said first genomic region fragments and at least one of said second genomic region fragments to create a ligation product;

d) purifying said ligation product with said affinity marker;

and

e) analyzing said ligation product under conditions such that a genomic interaction frequency for said ligation product is identified.

16. A method for identifying interaction frequencies, comprising:

a) crosslinking a nuclear matrix comprising a first genomic region and a second genomic region;

b) fragmenting said first genomic region and said second genomic region into a plurality of first genomic region fragments and a plurality of second genomic region fragments;

c) ligating a junction marker labeled with an affinity marker between at least one of said first genomic region fragments and at least one of said second genomic region fragments to create a ligation product;

d) purifying said ligation product with said affinity marker;

and

e) detecting a close proximity between said first genomic region and said second genomic region with said ligation product under conditions such that a genomic interaction frequency for said ligation product is identified.

17. A method for identifying interaction frequencies, comprising:

a) crosslinking a nuclear matrix comprising a first genomic region and a second genomic region;

b) fragmenting said first genomic region and said second genomic region into a plurality of first genomic region fragments and a plurality of second genomic region fragments;

c) ligating a junction marker labeled with an affinity marker between at least one of said first genomic region fragments and at least one of said second genomic region fragments to create a ligation product;

d) purifying said ligation product with said affinity marker;

and

e) identifying a contact probability as a function of genomic distance between said first genomic region and said second genomic region with said ligation product under conditions such that a genomic interaction frequency for said ligation product is identified.

18. A method for identifying interaction frequencies, comprising:

a) crosslinking a nuclear matrix comprising a first genomic region and a second genomic region;

b) fragmenting said first genomic region and said second genomic region into a plurality of first genomic region fragments and a plurality of second genomic region fragments;

c) ligating a junction marker labeled with an affinity marker between at least one of said first genomic region fragments and at least one of said second genomic region fragments to create a ligation product;

d) purifying said ligation product with said affinity marker;

and

e) identifying whether said first genomic region and said second genomic region preferentially associate with each other with said ligation product under conditions such that a genomic interaction frequency for said ligation product is identified.