Purification of mitochondrial and plastid DNA.
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.
Lang
B Franz
BF
Département de Biochimie, Robert Cedergren Center for Bioinformatics and Genomics, Canadian Institute for Advanced Research, Program in Evolutionary Biology, Université de Montréal, CP 6128, Montréal, Québec, Canada H3T 1J4. Franz.Lang@Umontreal.ca
Burger
Gertraud
G
D001482:Base Composition; D002498:Centrifugation; D002586:Cesium; D002712:Chlorides; D018742:DNA, Chloroplast / Q000302:isolation & purification*; D004272:DNA, Mitochondrial / Q000302:isolation & purification*; D005821:Genetic Techniques; D023281:Genomics / Q000379:methods*; D013045:Species Specificity
D002712:Chlorides; D018742:DNA, Chloroplast; D004272:DNA, Mitochondrial; D002586:Cesium; C028019:cesium chloride