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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*||30 mL|
|cccDNA Precipitation Solution*||100 mL|
*For a kit with only cccDNA Extraction Solution A, check out CAT#220501
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.
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.
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