Development and application of long-PCR for the assay of full-length animal mitochondrial DNA.

Native mitochondria1 (mt) DNA in higher animals exists as a closed-circular molecule typically 1&20 kilobase pairs (kb) in length. For nearly two decades, this cytoplasmically housed genetic system has been employed to estimate matrilineal phylogeny within and among species (Avise 1994). Beginning in the late-lWOs, a primary assay method involved restriction analyses of whole mtDNA molecules that had been physically isolated from nuclear DNA. In the late-l980s, many studies moved to PCR-based (Saiki et al. 1988) restriction-site or sequence assays of particular mtDNA regions typically a few hundred to lo00 bp in length. Here we document successful 'long-PCR amplification (Cohen 1994) of hll-length animal mtDNA, using cmserved 16s ribosomal gene sequences. Heretofore, PCR amplification of full-length mtDNA has been reported only for humans, and in a clinical context (Cheng et al. 1994b; Li et al. 1995). Assays were focused primarily on armadillos (Mammalia, Edentata, Dasypodidae): Dasypus norwncinctus (n = 12 specimens from scattered sites in the south-eastem United States); and Tolypeutes rnatacus ( n l), Zoedyus pichiy (n = 2), Chaetophrnctus wlIerOsus (n 2), and C. villosus (n = 2) collected in northern Argentina. From the nonDnsypus species, total genomic DNA was prepared from blood by standard methods involving phenol chloroform extraction and ethanol precipitation. For the other specimens, mtDNA was purified from heart and liver by conventional CsCl gradient centrifugation (Lansman et al. 1981). Total genomic DNA also was prepared from 20 additional specimens of Dasypus according to Taggart et nl. (1992), using clips of ear tissue preserved in 90% ethanol. Sequences (c. 500 bp long) within the mtDNA 16s ribosomal RNA gene were PCR-amplified using the 'universal' primers 16sar-L and 16sbr-H (Palumbi et al. 1991). Templates for these amplifications were CsCl gradient-