Abstract
We describe two simple protocols for preparation of templates for direct sequencing of yeast mitochondrial DNA (mtDNA) by automatic DNA analyzers. The protocols work with a range of yeast species and yield a sufficient quantity and quality of the template DNA. In combination with primer-walking strategy, they can be used either as an alternative or a complementary approach to shot-gun sequencing of random fragment DNA libraries. We demonstrate that the templates are suitable for re-sequencing of the mtDNA for comparative analyses of intraspecific variability of yeast strains as well as for primary determination of the complete mitochondrial genome sequence.
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Acknowledgments
The authors wish to thank L. Kovac (Department of Biochemistry, Comenius University, Bratislava) for continuous support, Sally A. Meyer (Georgia State University, Atlanta, USA), Paul F. Lehmann (Medical College of Ohio, Toledo, USA), and Cletus P. Kurtzman (National Center for Agricultural Utilization Research, Peoria, USA) for gifts of yeast strains, Geraldine Butler (Conway Institute, Dublin, Ireland) and Kenneth H. Wolfe (Smurfit Institute of Genetics, Dublin, Ireland) for the strain C. parapsilosis CLIB214 and the raw mtDNA sequence data from this strain, and Gabriel Minarik (DNA sequence facility, Comenius University, Bratislava, Slovakia) for excellent technical assistance. In addition, we would like to thank anonymous reviewer for her/his suggestions concerning the protocol employing total DNA preparation. This work was supported by grants from the Howard Hughes Medical Institute (55005622), the Fogarty International Research Collaboration Award (2-R03-TW005654-04A1), and the Slovak grant agencies VEGA (1/0219/08) and APVV (20-001604, LPP-0164-06).
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Communicated by C. Koehler.
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Valach, M., Tomaska, L. & Nosek, J. Preparation of yeast mitochondrial DNA for direct sequence analysis. Curr Genet 54, 105–109 (2008). https://doi.org/10.1007/s00294-008-0200-3
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DOI: https://doi.org/10.1007/s00294-008-0200-3