Genome-Wide Chromosome Conformation Capture

• US 20100081141A1
• Filed: 08/06/2009
• Published: 04/01/2010
• Est. Priority Date: 08/06/2008
• Status: Active Grant

• Current Assignee: University of Southern California
• Original Assignee: University of Southern California
• Inventors: Chen, Lin, Kalhor, Reza
• Granted Patent: US 8,076,070 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2565/531 characterised by the capture moiety being a protein for target oligonucleotides

Abstract

The invention relates to the use of GCC (Genome Conformation Capture) technology in determining the three dimensional arrangement of an entire genome.

Claims

1. A method of determining the three-dimensional arrangement of an entire genome in a cell, comprising:

(a) contacting a cell with a cross-linking reagent to cross-link the chromatin and the proteins in the cell;

(b) lysing the cell;

(c) digesting the chromatin with a restriction enzyme to generate DNA fragments with free ends, wherein the DNA fragments are cross-linked to the proteins;

(d) ligating DNA linkers to all free DNA ends generated by the restriction enzyme;

(e) annealing the ends of the DNA linkers that are not ligated to the free DNA ends generated by the restriction enzyme;

(f) translating the nicks between the annealed ends of the DNA linkers;

(g) detaching the proteins cross-linked to the DNA fragments;

(h) extracting DNA;

(i) subjecting the extracted DNA to two rounds of parallel sequencing; and

(j) determining the three-dimensional arrangement of the entire genome in the nucleus of the cell.

3. The method of claim 1, wherein the DNA linkers contain a palindromic overhang on one end and a T overhang on the other end.

4. The method of claim 3, wherein the DNA linkers are ligated to the free DNA ends generated by the restriction enzyme through the T overhang.

5. The method of claim 3, wherein the DNA linkers are ligated to the free DNA ends generated by the restriction enzyme by T4 DNA ligase.

6. The method of claim 1, further comprising adjusting the ligation mixture after step (d) to highly favor intramolecular reactions.

7. The method of claim 1, wherein the ends of the DNA linkers being annealed in step (e) are adjacent due to the DNA linkers being ligated to the DNA fragments that are cross-linked to the proteins.

8. The method of claim 1, wherein the nicks are translated with E. coli DNA polymerase I.

9. The method of claim 1, wherein the nicks are translated in both directions along the DNA fragments until they arrive at cross-linked sites.

10. The method of claim 1, further comprising adding phosphothioate-containing nucleotides to a second round of nick-translation.

11. The method of claim 1, wherein the proteins cross-linked to the DNA fragments are detached by heating at 65°

C. over night.

12. The method of claim 1, wherein the DNA is extracted with phenol:

chloroform.

13. The method of claim 1, further comprising removing DNA that has not gone through steps (d) and (e) and free DNA linkers after step (h).

14. The method of claim 13, wherein the DNA that has not gone through steps (d) and (e) and free DNA linkers are removed by a phosphothioate-sensitive exonuclease.

15. The method of claim 1, wherein the extracted DNA is sequenced using the Illumina/Solexa platform.

2. The method of clam 1, wherein the cross-linking reagent is formaldehyde.