Chromosome Conformation Analysis

• US 20120302449A1
• Filed: 05/18/2012
• Published: 11/29/2012
• Est. Priority Date: 05/18/2011
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Stevens, Junko F., Woo, Cora L., Montesclaros, Luz, Dong, Shoulian, Liu, Chunmei
• Granted Patent: US 8,663,919 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• 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 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2545/113 with an external standard/control, i.e. control reaction is separated from the test/target reaction
• CPC Class Codes: C12Q 2565/133 conformational analysis
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/178 miRNA, siRNA or ncRNA

Abstract

Disclosed herein are compositions, methods and kits for analyzing three-dimensional chromatin and/or chromosome conformation. Method are also disclosed for using the methods disclosed herein for diagnosing diseases such as cancer.

Claims

1. A method of determining chromosome conformation comprising:

a) isolating cells from a biological sample;

b) incubating the cells with a cross-linking agent, thereby cross-linking proteins with DNA and forming a cross-linked product;

c) lysing the cells;

d) digesting the DNA with a restriction endonuclease;

e) ligating the digested DNA thereby creating a ligation product;

f) reversing the cross-linking;

g) purifying the ligation product; and

h) analyzing the ligation product, whereby the chromatin arrangement is determined.

2. The method according to claim 1, wherein the cross-linking reagent comprises formaldehyde.

3. The method according to claim 1, further comprising the step of incubating the cross-linked product with a cross-linking quencher.

4. The method of claim 1, wherein the purifying step comprises

a) incubating the ligation product with magnetic beads;

b) collecting the ligation product bound to the magnetic beads; and

c) eluting the bound ligation product from the magnetic beads.

5. The method according to claim 1, wherein the lysing step comprises incubating the cells with one or more detergents.

6. The method according to claim 5, wherein the one or more detergents are selected from the group consisting of anionic detergents and non-ionic detergents.

7. The method according to claim 5, wherein the one or more detergents comprises a combination of one or more anionic detergents and one or more non-ionic detergents.

8. The method according to claim 1, wherein the analyzing step is performed using the polymerase chain reaction.

56. The method according to claim 1, further comprising a control assay to monitor undigested template DNA, wherein the control assay comprises:

a) a forward primer that hybridizes to a region in the template DNA upstream of the restriction endonuclease cutting site; and

b) a reverse primer that hybridizes to a region in the template DNA downstream of the restriction endonuclease cutting site.

57. The method according to claim 1, further comprising a control assay to monitor the digested template DNA, wherein the control assay comprises:

a) a bridge oligo comprising a blocked 3′

end, a template DNA binding region and a primer binding region;

b) a forward primer that hybridizes to a region in the 5′

end of the bridge oligo that is upstream of the restriction endonuclease cutting site;

c) a reverse primer that hybridizes to the template DNA that corresponds to a region downstream of the restriction endonuclease cutting site; and

d) optionally an oligonucleotide probe that anneals to a region containing the restriction endonuclease cutting site.

9-55. -55. (canceled)

58-68. -68. (canceled)