Abstract
The size, structure and conformation of mitochondrial and plastid genomes differ dramatically among eukaryotes. Similarly, the yield and purity of extracted organelle DNA also vary, and are crucial factors for the success of restriction mapping and sequencing experiments. We describe here procedures for the purification of organelle DNA from a broad range of eukaryotes. By emphasizing the underlying principles, these procedures will facilitate the development of new species-specific protocols. The presented purification schemes involve either isolation of organelles and subsequent extraction of DNA from this subcellular fraction, or processing of whole-cell lysates followed by CsCl gradient centrifugation to separate nuclear and organelle DNAs according to their A + T content. We have successfully used the described procedures for organelle genome sequencing from diverse eukaryotes, including non-axenic protists. Procedures can be completed in 3–5 days, typically yielding a few micrograms of DNA—ample for sequencing complete genomes.
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Acknowledgements
We thank Dr. M. Reith (Halifax) for providing us with his variant of CTAB DNA purification method that he optimized for red algae. This work was supported by the Canadian Institute of Health Research (B.F.L., grant MOP-42475; G.B., grant MOP-79309). Interaction support from the Canadian Institute of Advanced Research, Program in Evolutionary Biology (G.B. and B.F.L.) is gratefully acknowledged.
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Lang, B., Burger, G. Purification of mitochondrial and plastid DNA. Nat Protoc 2, 652–660 (2007). https://doi.org/10.1038/nprot.2007.58
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DOI: https://doi.org/10.1038/nprot.2007.58
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