Magnetic Capture Hybridization of Amphibian Mitochondrial Genomes
Abstract
Analysis of mitochondrial DNA (mtDNA) provides useful insights into population
dynamics of target organisms, and efficient extraction of this DNA is pivotal to ensuring
the reliability of subsequent reactions. When extracted from organic tissue, resulting
solutions contain a mixture of genomic and mitochondrial DNA, and separation of these
two molecules usually requires the use of an ultracentrifuge; because many amphibian
organisms of interest are found in tropical regions of the world without ready access to
the required machinery, a new, more portable, method of selectively isolating
mitochondrial genomes from complex mixtures to facilitate whole mitochondrial genome
sequencing is proposed here. Conserved sequences in the 12S and 16S regions can be
targeted by a uniquely designed biotinylated probe that will form a complex with
magnetic beads covalently coupled to streptavidin. This research shows that the complex
can be selectively filtered from solution through magnetic capture, and heat treatment
then dissociates the probe complex from the mtDNA. The resulting solution contains
samples suitable for downstream reactions to expand the knowledge of population
dynamics as they relate to the emerging threat of chytridiomycosis in Anuran
populations.