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Circle-Seq: Isolation and Sequencing of Chromosome-Derived Circular DNA Elements in Cells

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  • Henrik Devitt Møller

Chromosome-derived extrachromosomal circular DNA elements (eccDNAs) are detected in all eukaryotes examined so far. Here I describe the Circle-Seq protocol, applicable for physical enrichment of eccDNAs of a broad size range, combined with sequence confirmation of circular structures.Briefly, by concise alkaline treatment and gentle gravity flow-through an ion-exchange column, eccDNAs are enriched in the eluate fraction. EccDNAs are enzymatically isolated by extensive Plasmid-Safe DNase digestion of linear chromosomes and further enriched by φ29 rolling circle amplification. By means of high throughput sequencing of amplified eccDNA and custom eccDNA mapping software, around ten-thousand unique eccDNA types could be detected at nucleotide resolution in a million human muscle nuclei by this method.

OriginalsprogEngelsk
TitelDNA Electrophoresis : Methods and Protocols
RedaktørerKatsuhiro Hanada
Antal sider17
ForlagHumana Press
Publikationsdato2020
Sider165-181
Kapitel15
ISBN (Trykt) 978-1-0716-0322-2
ISBN (Elektronisk)978-1-0716-0323-9
DOI
StatusUdgivet - 2020
NavnMethods in Molecular Biology
Vol/bind2119
ISSN1064-3745

ID: 235588304