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Cell Biology JoVE (Open Access) Citable · DOI

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

DOI: 10.3791/54239-v
What you'll learn
  • ✓ Purify extrachromosomal circular DNA from eukaryotic cells using Circle-Seq
  • ✓ Remove linear chromosomal DNA and amplify eccDNA via rolling-circle amplification
  • ✓ Prepare eccDNA samples for high-throughput sequencing and genome-wide analysis
  • ✓ Identify and quantify copy-number variations and genome instability markers
Protocol

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

Difficulty
advanced
Total time
~4–6 hours per sample (excluding sequencing run time)
Biosafety
BSL-1

Steps

1
Harvest and prepare eukaryotic cells

Collect cultured eukaryotic cells and prepare them for extrachromosomal DNA extraction. This establishes the starting material for downstream purification.

▶ 01:07
2
Perform column purification of total DNA

Use column-based extraction to isolate total DNA from cell lysates, removing proteins and other cellular contaminants.

▶ 01:49
3
Digest linear chromosomal DNA selectively

Treat the DNA sample with exonucleases to remove linear chromosomal DNA while preserving circular eccDNA molecules.

▶ 04:03
4
Amplify circular DNA via rolling-circle amplification

Perform rolling-circle amplification on purified eccDNA to generate sufficient DNA for downstream sequencing library preparation.

▶ 06:16
5
Prepare amplified DNA for high-throughput sequencing

Process amplified eccDNA and prepare sequencing libraries for genome-wide analysis and data acquisition.

▶ 07:06
6
Analyze representative eccDNA sequencing results

Examine and interpret Circle-Seq output data, including eccDNA abundance, genomic distribution, and copy-number variation patterns.

▶ 07:53
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