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.