Sequencing complete mitochondrial and plastid genomes.
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.
Burger
Gertraud
G
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@UMontreal.CA
Lavrov
Dennis V
DV
Forget
Lise
L
Lang
B Franz
BF
D003001:Cloning, Molecular / Q000379:methods; D004272:DNA, Mitochondrial / Q000235:genetics*; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D023281:Genomics / Q000379:methods*; D018087:Plastids / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
D004272:DNA, Mitochondrial