Comparison of Mitochodrial DNA in Wolves and Coyotes in the Northern Rockies using the Polymerase Chain Reaction Technique

Abstract

Conservation of biological species, often involves the introduction of organisms from one population to a site in which a population has gone locally extinct. The genetic constitution of the introduced organisms is of immediate concern both in terms of restoring the original population as nearly as possible and to maintain genetic diversity of the introduced organisms. Molecular techniques using protein or isozyme variation and DNA Restriction Fragment Length Polymorphisms (RFLPs), have been used to estimate genetic variation. These techniques are not sensitive enough to make comparisons using limited sample sizes or to analyze samples from preserved specimens of extinct organisms. The advent of the Polymerase Chain Reaction (PCR) (Saiki et al. 1985, 1988) which amplifies small segments of DNA millions of times has extended the application of molecular biology techniques to the genetic comparisons of dried or alcohol preserved museum specimens to extant organisms (Paabo 1989 and Paabo et al. 1989). Application of this technique has allowed the comparison of extinct organisms to each other and to extant species (Thomas et al. 1990). PCR synthesizes many copies of the target sequence greatly increasing the quantity of the amplified sequence. PCR involves denaturing or strand separation of the DNA, hybridization of primers to the denatured single strands and then enzymatic extension of the primers using strands of the sample DNA as a template. This cycle is repeated many times, theoretically amplyfying the target DNA twofold with each cycle. Therefore, after n cycles there is 2 to the nth power as much of the target sequence DNA as there was in the original sample. Essentials of the PCR technique are shown in Figures 1 and 2 taken from Arnheim et al. (1990).