Rapid isolation of animal mitochondrial DNA in a small fixed-angle rotor at ultrahigh speed.

Abstract

In recent years, mitochondrial DNA (mtDNA) sequence analysis has become a powerful tool for the investigation of the evolutionary and population biology of various animal species (Wilson et al., 1985). Most such studies have relied on physical purification of mtDNA, typically by ultracentrifugation in cesium chloride gradients. Thorough descriptions of such methods are given by Lansman et al. (1981) and Wright et al. (1983). Practical concerns over the expensive instrumentation and extensive time required by such methods have been expressed by several workers (e.g., Grill et al., 1983; Powell and Zufiiga, 1983). Several of our colleagues have expressed doubt that purified mtDNA samples can be generated in a reasonable time in numbers sufficient to perform large-scale population studies. We describe here an ultracentrifugal procedure that yields homogeneous mitochondrial DNA from as many as i0 samples in as little as 10 hr. The procedure uses a relatively inexpensive tabletop ultracentrifuge and relies on a self-forming cesium chloride gradient generated in a small fixed-angle rotor that operates at a speed of 100,000 rpm and a relative centrifugal force of 436,000g.