Purification and Sequencing of Extrachromosomal DNA

UACC-1598–4 cell line was alkaline treated to separate chromosomal DNA, lipids, and protein from eccDNA by rapid DNA denaturing–renaturing, followed by column chromatography on an ion exchange membrane column (Plasmid Mini AX; A&A Biotechnology), The eccDNA was purified according to the purification method provided in the document, linear and mitochondrial DNA were removed by endonucleases and rolling-circle amplification of eccDNA by Phi29 polymerase reactions (REPLI-g Midi Kit) amplifying DNA at 30 °C for 2 days for Circle-Seq and then the eccDNA was sequenced based on Illumina platform [17 (link)].

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Zhang Y., Dong K., Jia X., Du S., Wang D., Wang L., Qu H., Zhu S., Wang Y., Wang Z., Zhang S., Sun W, & Fu S. (2023). A novel extrachromosomal circular DNA related genes signature for overall survival prediction in patients with ovarian cancer. BMC Medical Genomics, 16, 140.

Publication 2023

Plasmid Mini AX

Cell line Chromatography Chromosomal Endonucleases Ion exchange Lipids Membrane Mitochondrial dna Plasmid Protein

Corresponding Organization :

Other organizations : Harbin Medical University, Second Affiliated Hospital of Harbin Medical University, Third Affiliated Hospital of Harbin Medical University

Top 5 similar protocols

Variable analysis

independent variables

Alkaline treatment to separate chromosomal DNA, lipids, and protein from eccDNA
Rapid DNA denaturing–renaturing
Column chromatography on an ion exchange membrane column (Plasmid Mini AX; A&A Biotechnology)
Removal of linear and mitochondrial DNA by endonucleases
Rolling-circle amplification of eccDNA by Phi29 polymerase reactions (REPLI-g Midi Kit)

dependent variables

Purification of eccDNA
Sequencing of eccDNA based on Illumina platform

control variables

Amplification of eccDNA at 30 °C for 2 days for Circle-Seq

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