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Covalently closed circular DNA (cccDNA) is the natural form of most plasmids, it is also a key replicative intermediate in the life cycles of some viruses like HBV. In higher organisms including human, cccDNA is called extrachromosomal circular DNA (eccDNA)，which play a role in eliciting innate immune responses, mediating cell-to-cell communication, advancing genetic heterogeneity, compensating for genetic loss, aging, regulating gene expression and molecular sponging. In addition, cell-free eccDNAs in circulating system could be used as novel biomarkers for the diagnosis and prognosis of cancer.
(1) The isolation of cccDNA from non-cccDNA is crucial to study cccDNA. Various isolation methods have been reported. Several techniques exploit the structural differences between chromosomal and circular DNA and separate them by SDS lysis-salt precipitation (2) or high-speed ultracentrifugation in cesium-chloride gradients (3). Most methods encompass extraction of total DNA purification followed by several rounds of enzymatic elimination of non-cccDNA and a final step of cccDNA extractions. The enzyme used includes exonuclease III which digest linear and open circular forms of DNA (4) , Plasmid-Safe DNase (5 -7) which preferentially hydrolyzes double-stranded linear DNA (dslDNA) and, with a lower efficiency, linear and closed circular single-stranded DNAs (ssDNAs), a combination of exonuclease I and exonuclease III which degrade DNA strands with free 3′ ends (8),and T5 Exonuclease (9),which specifically digest nicked double-stranded circular DNA. This product is designed to purify cccDNAs from total DNA samples or partially purified cccDNA samples.
- One step of extraction, simple to perform.
- Minimal non-cccDNA contamination.
- Maximal cccDNA recovery rate.
- No enzymatic digestion.
- Applicable to cccDNA of any size.
- Compatible with silica-based purification (in both column format and magnetic beads format).
- Enough for 100 times of purification.
- Research use only.
|cccDNA Extraction Solution A||100 mL|
|cccDNA Extraction Solution B*||N/A|
|cccDNA Precipitation Solution*||N/A|
*cccDNA Extraction Solution B and cccDNA Precipitation Solution are included exclusively with CAT#220502
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.
References:Man Wang, et al. 2021. Extrachromosomal Circular DNAs: Origin, formation and emerging function in Cancer. International Journal of Biological Sciences 17(4): 1010-1025
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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.
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