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Updated: Jul 3, 2026
14:26
Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells
Published on: April 4, 2016
Method to eliminate linear DNA from mixture containing nicked circular, supercoiled, and linear plasmid DNA
Pichumani Balagurumoorthy1, S James Adelstein, Amin I Kassis
1Department of Radiology, Harvard Medical School, Boston, MA 02115, USA. pbalagurumoorthy@hms.harvard.edu
Analytical Biochemistry
|July 22, 2008
Summary
This study introduces a novel enzymatic method using lambda exonuclease and RecJ(f) to efficiently remove linear DNA contaminants. This technique purifies circular plasmid DNA, crucial for therapeutic and biophysical applications.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Circular plasmid DNA preparations frequently contain linear DNA fragments.
- These contaminants originate from DNA degradation or linearization.
- Linear DNA hinders applications requiring pure circular plasmid DNA.
Purpose of the Study:
- To develop a selective enzymatic method for removing linear DNA from plasmid preparations.
- To ensure the integrity of circular plasmid DNA (supercoiled and nicked) during purification.
Main Methods:
- Utilized a combination of lambda exonuclease and RecJ(f) (a single-strand-specific exonuclease).
- Lambda exonuclease digests one strand of linear DNA (5' to 3').
- RecJ(f) digests the remaining complementary strand, eliminating linear DNA.
Main Results:
- The enzymatic treatment effectively removed linear DNA from mixtures containing supercoiled DNA.
- Supercoiled circular DNA remained intact after the enzymatic process.
- The method also successfully removed linear DNA from preparations of nicked circular DNA.
Conclusions:
- A simple and effective enzymatic method for purifying circular plasmid DNA is presented.
- This technique is valuable for preparing homogeneous circular plasmid DNA for therapeutic and biophysical studies.
- The enzyme combination selectively targets linear DNA without affecting circular forms.
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