Genome-wide purification of extrachromosomal circular DNA from eukaryotic cells

Henrik D. Møller; Rasmus Kenneth Bojsen; Chris Tachibana; Lance Parsons; David Botstein; Birgitte Regenberg

doi:10.3791/54239

Genome-wide purification of extrachromosomal circular DNA from eukaryotic cells

Henrik D. Møller, Rasmus Kenneth Bojsen, Chris Tachibana, Lance Parsons, David Botstein, Birgitte Regenberg

15 Citationer (Scopus)

64 Downloads (Pure)

Abstract

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are found in other eukaryotes. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and environmental and biological factors that induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, high-throughput sequencing, and mapping. Extensive exonuclease treatment (more than 100 units) was typically required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by #29 polymerase enriched for circular DNA over linear DNA. Validation of theCircle-Seq method on three S. cerevisiae CEN.PK populations of 10¹⁰ cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

Originalsprog	Engelsk
Artikelnummer	e54239
Tidsskrift	Journal of Visualized Experiments
Udgave nummer	110
Antal sider	8
ISSN	1940-087X
DOI	https://doi.org/10.3791/54239
Status	Udgivet - 12 feb. 2016

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10.3791/54239Licens: CC BY-NC-ND

Møller_2016_Genome-wide_PurificationForlagets udgivne version, 660 KBLicens: CC BY-NC-ND

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abstract = "Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are found in other eukaryotes. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and environmental and biological factors that induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, high-throughput sequencing, and mapping. Extensive exonuclease treatment (more than 100 units) was typically required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by #29 polymerase enriched for circular DNA over linear DNA. Validation of theCircle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.",

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AU - Møller, Henrik D.

AU - Bojsen, Rasmus Kenneth

AU - Tachibana, Chris

AU - Parsons, Lance

AU - Botstein, David

AU - Regenberg, Birgitte

PY - 2016/2/12

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N2 - Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are found in other eukaryotes. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and environmental and biological factors that induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, high-throughput sequencing, and mapping. Extensive exonuclease treatment (more than 100 units) was typically required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by #29 polymerase enriched for circular DNA over linear DNA. Validation of theCircle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

AB - Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are found in other eukaryotes. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and environmental and biological factors that induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, high-throughput sequencing, and mapping. Extensive exonuclease treatment (more than 100 units) was typically required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by #29 polymerase enriched for circular DNA over linear DNA. Validation of theCircle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

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DO - 10.3791/54239

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SN - 1940-087X

JO - Journal of Visualized Experiments

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Genome-wide purification of extrachromosomal circular DNA from eukaryotic cells

Abstract

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