Purification and Sequencing of Large Circular DNA from Human Cells
This method is adapted from Møller et. al. (2015)1 and optimized to extract large circular DNA from human cancer cells. Previous methods of isolating circular DNA species have involved CsCl-EtBr density gradients and 2D gel electrophoresis of genomic DNA2. While Møller et. al. (2015) use column purification to isolate circular DNA from yeast, our protocol uses magnetic bead-based purification to extract low and high molecular weight circular DNA from human cells. Although the biological significance of circular DNA is incompletely understood, recent work has shown that it is prevalent in yeast1 and cancer cells3, has demonstrated that circular DNA amplification can drive intratumoral heterogeneity3, and has shown that circular DNA may form via microdeletions4 and intrachromatid homologous recombination5.
This is a list of supplementary files associated with this preprint. Click to download.
Protocol as PDF Protocol as PDF This is the entire protocol in PDF format for download.
Posted 22 Jan, 2019
Purification and Sequencing of Large Circular DNA from Human Cells
Posted 22 Jan, 2019
This method is adapted from Møller et. al. (2015)1 and optimized to extract large circular DNA from human cancer cells. Previous methods of isolating circular DNA species have involved CsCl-EtBr density gradients and 2D gel electrophoresis of genomic DNA2. While Møller et. al. (2015) use column purification to isolate circular DNA from yeast, our protocol uses magnetic bead-based purification to extract low and high molecular weight circular DNA from human cells. Although the biological significance of circular DNA is incompletely understood, recent work has shown that it is prevalent in yeast1 and cancer cells3, has demonstrated that circular DNA amplification can drive intratumoral heterogeneity3, and has shown that circular DNA may form via microdeletions4 and intrachromatid homologous recombination5.
© Research Square 2021