Fast, non-enzymatic isolation of covalently closed circular DNA (cccDNA) from total DNA samples. Ideal for HBV studies, circulating eccDNA profiling, and cancer biomarker research. 100 preps/kit. For research use only.

Description

Covalently closed circular DNA (cccDNA) is the natural form of most plasmids. It is also a key replicative intermediate in the life cycles of certain viruses, such as hepatitis B virus (HBV). In higher organisms, including humans, cccDNA-like structures are referred to as extrachromosomal circular DNA (eccDNA). These molecules have been shown to play roles in eliciting innate immune responses, mediating cell-to-cell communication, promoting genetic heterogeneity, compensating for genetic loss, contributing to aging, regulating gene expression, and functioning as molecular sponges. Additionally, cell-free eccDNAs circulating in bodily fluids have emerged as potential biomarkers for the diagnosis and prognosis of various cancers.

The isolation of cccDNA from non-cccDNA is critical for studying its biological functions. Various isolation methods have been reported. Several techniques exploit the structural differences between chromosomal and circular DNA, enabling their separation via SDS lysis–salt precipitation or high-speed ultracentrifugation in cesium chloride gradients.

Most protocols involve total DNA extraction followed by multiple rounds of enzymatic digestion to eliminate non-cccDNA, and a final step of cccDNA purification. Enzymes used include:

Exonuclease III, which digests linear and open circular forms of DNA
Plasmid-Safe DNase, which preferentially hydrolyzes double-stranded linear DNA (dslDNA), and to a lesser extent, linear and closed circular single-stranded DNA (ssDNA)
A combination of Exonuclease I and Exonuclease III, which degrade DNA strands with free 3′ ends
T5 Exonuclease, which specifically digests nicked double-stranded circular DNA

This product is designed to purify cccDNA from either total DNA samples or partially purified preparations, offering a streamlined, enzyme-free workflow.

Features

No Enzymatic Digestion Required — avoids DNA degradation from residual nucleases
One-Step Extraction Protocol — time-saving and user-friendly
High cccDNA Recovery Efficiency — minimal contamination with non-circular forms
Broad Compatibility — suitable for cccDNA of all sizes
Flexible Format — compatible with silica-based columns and magnetic bead systems
100 Preparations per Kit
For Research Use Only

Applications

HBV Research: Isolation of HBV cccDNA from infected liver tissues or cell models
Cancer Biomarker Discovery: Extraction of circulating eccDNA from plasma for diagnostic/prognostic research
Aging and Genetic Heterogeneity Studies: eccDNA profiling in aging models and cancer cell lines
Gene Expression and Molecular Sponging Research
High-throughput DNA Structure Analysis

Kit Contents

Component	CAT#:220501
cccDNA Extraction Solution A	100 mL
cccDNA Extraction Solution B*	N/A
cccDNA Precipitation Solution*	N/A

Note: For greater convenience, cccDNA Extraction Solution B and cccDNA Precipitation Solution are included in the extended version (CAT# 220502), eliminating the need to prepare additional reagents separately.

Input Sample Requirements

Highly suggest to use SDS-proteinase K digestion and alcohol precipitation method to isolated total DNA since proteinase K can release cccDNA from possible covalent link to proteins. In addition, alcohol precipitation can recover non-exclusively both non-cccDNA and cccDNA.
To avoid the nicking of cccDNA by residual DNase in total DNA preparation, perform cccDNA purification immediately after total DNA isolation, or quick-freeze total DNA preparation immediately and store at –80°C until cccDNA isolation.
Total DNA should be dissolved in DNase-free water or TE solution, not in other solvent, otherwise a trial experiment is needed to test the compatibility of this solvent and this kit.

Storage and Handling

Store the kit at 4 °C. The kit is stable for at least one year from date of receipt.

User Manual:

Click here for more guides and user manual.

References

Yuangao Wang, et al. Purification, full-length sequencing and gemonic origin mapping of eccDNA, 2022 Nature Protocals

Yuangao Wang, et al. eccDNAs are apoptotic products with high innate immunostimulatory activity. 2021 Nature

Man Wang, et al. 2021. Extrachromosomal Circular DNAs: Origin, formation and emerging function in Cancer. International Journal of Biological Sciences 17(4): 1010-1025

Hirt B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 26:365–369.

van Loon N, Miller D, Murnane JP. 1994. Formation of extrachromosomal circular DNA in HeLa cells by nonhomologous recombination. Nucleic Acids Research:22(13):2447–2452.

James W. Gaubatz, 1990. Purification of eucaryotic extrachromosomal circular DNAs using exonuclease III. Analytical Biochemistry 184(2) : 305-310

Werle-Lapostolle B, et.al. 2004. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology 126:1750–1758

Kock J, et al. 2010. Generation of covalently closed circular DNA of hepatitis B viruses via intracellular recycling is regulated in a virus specific manner. PLoS Pathog. 6:e1001082

Schnepp BC, et al. 2003. Genetic fate of recombinant adeno-associated virus vector genomes in muscle. J. Virol. 77:3495–3504

Luo J, Cui X, Gao L, Hu J. 2017. Identification of an intermediate in hepatitis B virus covalently closed circular (ccc) DNA formation and sensitive and selective cccDNA detection. J. Virol. 91:e00539-17).

Bingqian Qu,et al. 2018. T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR. Virol.92(23) :e01117-18

Vinograd J, Lebowitz J. 1966. Physical and Topological Properties of Circular DNA. Journal of General Physiology. 49(6P2):103.

Shibata Y, Kumar P, et al. 2012. Extrachromosomal MicroDNAs and Chromosomal Microdeletions in Normal Tissues. Science. 336(6077): 82–86.

cccDNA Purification Kit (100)-220501