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Sequencing complete mitochondrial and plastid genomes

Nature Protocols volume 2, pages 603–614 (2007)Cite this article

Abstract

Organelle genomics has become an increasingly important research field, with applications in molecular modeling, phylogeny, taxonomy, population genetics and biodiversity. Typically, research projects involve the determination and comparative analysis of complete mitochondrial and plastid genome sequences, either from closely related species or from a taxonomically broad range of organisms. Here, we describe two alternative organelle genome sequencing protocols. The “random genome sequencing” protocol is suited for the large majority of organelle genomes irrespective of their size. It involves DNA fragmentation by shearing (nebulization) and blunt-end cloning of the resulting fragments into pUC or BlueScript-type vectors. This protocol excels in randomness of clone libraries as well as in time and cost-effectiveness. The “long-PCR-based genome sequencing” protocol is specifically adapted for DNAs of low purity and quantity, and is particularly effective for small organelle genomes. Library construction by either protocol can be completed within 1 week.

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Figure 1
Figure 2: PCR steps involved in the long-PCR-based genome sequencing protocol (protocol option B).
Figure 3: Nebulized and size-separated orgDNA.

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Acknowledgements

We thank Shona Teijeiro for critical reading of the manuscript and for help in the preparation of the figures. This work was supported by the Canadian Institute of Health Research (B.F.L., grant MOP-42475; G.B., grant MOP-79309; postdoctoral fellowship to D.V.L.). 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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  1. Dennis V Lavrov

    Present address: Present address: Department of Ecology, Evolution and Organismal Biology, Iowa State University, Ames, Iowa 50011, USA.,

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  1. Département de Biochimie, Robert Cedergren Centre, Program in Evolutionary Biology, Canadian Institute for Advanced Research, Université de Montréal, 2900 Boulevard Edouard-Montpetit, CP 6128, Montréal, Québec, H3T 1J4, Canada

    Gertraud Burger, Dennis V Lavrov, Lise Forget & B Franz Lang

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Correspondence to Gertraud Burger or B Franz Lang.

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Burger, G., Lavrov, D., Forget, L. et al. Sequencing complete mitochondrial and plastid genomes. Nat Protoc 2, 603–614 (2007). https://doi.org/10.1038/nprot.2007.59

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