Home › Analytical Chem › Associated Chromosome Trap for Identifying Long-range DNA Interactions
Analytical Chem JoVE (Open Access) Citable · DOI

Associated Chromosome Trap for Identifying Long-range DNA Interactions

DOI: 10.3791/2621-v
What you'll learn
  • ✓ Fix long-range chromatin interactions with formaldehyde
  • ✓ Isolate nuclei and digest chromatin for ligation
  • ✓ Ligate and purify interaction DNA segments
  • ✓ Amplify and sequence ACT libraries
Protocol

Biopharma Insights The associated chromosome trap (ACT) assay is a novel unbiased method for identifying long-range DNA interactions. The characterization of long range DNA interactions will allow us to determine the relationship of nuclear architecture to gene expression in both normal physiology and in diseased states.

Difficulty
advanced
Total time
~3-4 days
Biosafety
BSL-1

Steps

1
Fix chromatin interactions and isolate nuclei

Crosslink long-range DNA interactions with formaldehyde and isolate nuclei for downstream processing.

▶ 01:30
2
Digest, ligate, and purify interaction DNA

Digest chromatin and perform ligation to capture associated DNA segments, then purify the products.

▶ 03:36
3
Digest with Msp I and ligate linkers

Perform secondary digestion with Msp I and ligate oligonucleotide linkers to DNA ends.

▶ 06:10
4
Amplify and sequence ACT libraries

PCR amplify the ligated products and analyze sequences to identify interacting regions.

▶ 07:50
5
Review representative ACT assay results

Examine typical results from the ACT assay to interpret long-range DNA interactions.

▶ 11:06
6
Conclude and summarize ACT method

Summarize the ACT assay workflow and its applications in studying nuclear architecture.

▶ 14:23
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