Evolution Dynamics of mitochondrial DNA evolution in animals : Amplification and sequencing with conserved primers ( cytochrome b / 12 S ribosomal DNA / control region / evolutionary genetics / molecular phylogenies )

With a standard set of primers directed toward conserved regions, we have used the polymerase chain reaction to amplify homologous segments ofmtDNA from more than 100 animal species, including mammals, birds, amphibians, fishes, and some invertebrates. Amplification and direct sequencing were possible using unpurified mtDNA from nanogram samples of fresh specimens and microgram amounts of tissues preserved for months in alcohol or decades in the dry state. The bird and fish sequences evolve with the same strong bias toward transitions that holds for mammals. However, because the light strand of birds is deficient in thymine, thymine to cytosine transitions are less common than in other taxa. Amino acid replacement in a segment of the cytochrome b gene is faster in mammals and birds than in fishes and the pattern of replacements fits the structural hypothesis for cytochrome b. The unexpectedly wide taxonomic utility ofthese primers offers opportunities for phylogenetic and population research. During the past decade, geneticists and taxonomists have used restriction endonucleases rather than sequencing to examine variation within and between species in specific segments ofDNA (1-7). Although the indirect assessment of sequence variation obtained with the restriction endonuclease method is known to have many drawbacks,§ sequence data have been difficult to obtain. The construction and screening of clone libraries has been too tedious and have demanded too much expertise for routine use by those geneticists and taxonomists who must analyze many individuals. Dependence on restriction analysis has limited our understanding of the dynamics of DNA sequence evolution. The presence or absence of a restriction site reveals little about the kinds of nucleotide substitutions that have occurred. Thus, although restriction analysis of mtDNA from closely related mammals first showed that these genomes have a higher rate of evolutionary substitution than does nuclear DNA, the demonstration that this acceleration results mainly from an increase in the number of transitions relative to transversions came only from conventional cloning and sequencing (1, 3). Because most studies of animal mtDNA have used restriction analysis, it has been difficult to determine whether a high rate of evolution and a transition bias are characteristic of all animal mtDNAs (8-10). There has been a need for simple methods of sequencing mtDNA to examine the pattern of evolutionary substitution in other animal groups. A fast alternative to conventional cloning has emerged in the form of the polymerase chain reaction (11). By using this method, unique sequences can be cloned in vitro in a matter of hours. Furthermore, the procedure is easily automated, so that hundreds of samples can be amplified each day. The enzymatic amplification of a specific DNA segment is made possible by the highly specific binding of oligonucleotide primers to sequences flanking the segment. These primers allow the binding of a DNA polymerase that then copies the segment. Because each newly made copy can serve as a template for further duplication, the number of copies of the target segment grows exponentially (12). Wrischnik et al. (13) found an easy way to sequence the product of this reaction directly. The direct sequencing approach has since been improved (14) and applied to the study of mtDNA variation within the human species (13, 15-17). Because mtDNA differences among animal species are large (3), it was not expected that primers that amplified specific segments of human mtDNA would amplify the corresponding segments of mtDNA from other species. Nonetheless, we now describe three pairs of primers that amplify homologous sequences from a wide array of animals. This innovation has allowed us to gather sequence data$ to evaluate the pattern of molecular evolution in a variety of animal species. MATERIALS AND METHODS Primers. To design these primers, we compared the published sequences for mammal (18-20), frog (21), and fly (22) mtDNAs and searched for highly conserved regions. The sequences of the seven primers follow, the letters L and H refer to the light and heavy strands, and the number refers to the position of the 3' base of the primer in the complete human mtDNA sequence (18): cytochrome b, L14841 (5'-AAAAAGCTTCCATCCAACATCTCAGCATGATGAAA-3') and H15149 (5'-AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-3'); 12S rRNA, L1091 (5'-AAAAAGCTTCAAACTGGGATTAGATACCCCACTAT-3') and H1478 (5'-TGACTGCAGAGGGTGACGGGCGGTGTGT3'); control region, L15926 (5'-TCAAAGCTTACACCAGTCTTGTAAACC-3'), L16007 (5'-CCCAAAGCTAAAATTCTAA-3'), and H00651 (5'-TAACTGCAGAAGGCTAGGACCAAACCT-3'). §The preparation and alignment of restriction maps is itself tedious and subject to errors. Restriction sites offer a biased sample of nucleotide sequences. It is hard to compare results from different laboratories because of variation in the gel separation techniques and restriction enzymes used. When applied to mtDNA, the restriction method is reliable only among closely related taxa. Restriction analysis is limited to the small proportion of species in the biosphere that are big enough to provide milligram amounts of tissue or that can be cultured. The traces of short, modified DNA that survive in museum specimens or ancient remains cannot be analyzed with restriction enzymes. $The sequences reported in this paper have been deposited in the GenBank data base (accession nos. M25681-M25695). 6196 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 86 (1989) 6197 Many such priming regions exist, so that it is possible to amplify almost any segment of a mtDNA genome at will. In choosing oligonucleotide sequences, we took advantage of the evolutionary stability of regions of rRNA, the anticodon loops of tRNAs, and the active sites of enzymes. The 3' ends of primers were located on the first or second base of codons for amino acids that are evolutionarily conserved (e.g., tryptophan). Even primers with several mismatches to the template can be used for amplification; the polymerase requires absolute matching of the primer to the template only in the last few bases of the 3' end of the oligonucleotide. DNA Extraction. DNA was extracted from tissues by digestion in 100mM Tris HCI, pH 8.0/10mM EDTA/100 mM NaCI/0.1% SDS/50 mM dithiothreitol/proteinase K (0.5 gg/ml) for 2-4 hr at 37TC. The DNA was purified by extracting twice with phenol, once with phenol/chloroform [1:1 (vol/vol)], and once with chloroform. The sample was then concentrated by centrifugal dialysis (Centricon-30, Amicon) or ethanol precipitation. Polymerase Chain Reaction. Amplification was performed in 100 1.l ofa solution containing 67mM Tris (pH 8.8), 6.7mM MgSO4, 16.6 mM (NH4)2SO4, 10 mM 2-mercaptoethanol, each dNTP at 1 mM, each primer at 1 ,uM, genomic DNA (10-1000 ng), and 2-5 units of Thermus aquaticus polymerase (Perkin-Elmer/Cetus). Each cycle of the polymerase chain reaction consisted of denaturation for 1 min at 930C, hybridization for 1 min at 50'C, and extension for 2-5 min at 720C. This cycle was repeated 25-40 times depending on the initial concentration of template DNA in the sample. Generation of Single-Stranded DNA and Sequencing. Electrophoresis of 5 gl of the amplified mixture was done in a 2% agarose gel (NuSieve, FMC) in 40 mM Tris acetate (pH 8.0) and the DNA was stained with ethidium bromide. The gel fragment containing the amplified product was excised from the gel and melted in 1 ml of distilled water, and 1 . ul of this mixture was used as the template in a second chain reaction to generate single-stranded DNA for sequencing (14). In this second reaction, the concentration ofone or the other primer was reduced 100-fold. After 40 cycles of amplification, free nucleotides and salts were removed by 2-4 cycles of centrifugal dialysis. The DNA was sequenced with a commercial kit (Sequenase, United States Biochemical) and the primer that had been limiting in the second chain reaction.