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Molecular Phylogenetics and EvolutionVol. 12, No. 2, July, pp. 105–114, 1999Article ID mpev.1998.0602, available online at http://www.idealibrary.com on

Primers for a PCR-Based Approach to Mitochondrial GenomeSequencing in Birds and Other Vertebrates

Michael D. Sorenson,*,1 Jennifer C. Ast,† Derek E. Dimcheff,† Tamaki Yuri,†and David P. Mindell†

*Department of Biology, Boston University, Boston, Massachusetts 02215; and †Museum of Zoology and Department of Biology,University of Michigan, Ann Arbor, Michigan 48109-1079

Received June 24, 1998; revised September 24, 1998

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A PCR-based approach to sequencing complete mito-hondrial genomes is described along with a set of 86rimers designed primarily for avian mitochondrialNA (mtDNA). This PCR-based approach allows anccurate determination of complete mtDNA sequenceshat is faster than sequencing cloned mtDNA. Therimers are spaced at about 500-base intervals alongoth DNA strands. Many of the primers incorporateegenerate positions to accommodate variation intDNA sequence among avian taxa and to reduce the

otential for preferential amplification of nuclear pseu-ogenes. Comparison with published vertebratetDNA sequences suggests that many of the primersill have broad taxonomic utility. In addition, theserimers should make available a wider variety ofitochondrial genes for studies based on smaller data

ets. r 1999 Academic Press

Because of its maternal inheritance, haploidy, andapid rate of evolution, mitochondrial DNA (mtDNA)as many advantages as a marker for phylogeneticnalyses (Moore, 1995) and is one of the most fre-uently used markers in molecular systematics (e.g.,indell, 1997). While advances in DNA sequencing

echnology have made it possible to determine longerequences for larger samples of taxa, we believe thathe sampling of mitochondrial genes for systematictudies continues to be limited by the availability ofolymerase chain reaction (PCR) primers. In aviantudies, the cytochrome b gene has been sequencedore often than all other mitochondrial genes com-

ined (Table 1), presumably due to the early availabil-ty of ‘‘universal’’ primers for this gene (Kocher et al.,989). This historical inertia should be replaced by aeliberate choice of the genes that are likely to be theost informative for a given study. In addition, it now

1 To whom correspondence should be addressed at Department ofiology, Boston University, 5 Cummington Street, Boston, MA 02215.

sax: (617) 353-6340. E-mail: msoren@bu.edu.

105

ppears that adequate resolution of higher-level rela-ionships in birds and other taxa will require largerata sets, including a number of different genes (Cum-ings et al., 1995; Mindell et al., 1997) or even the

ntire mitochondrial genome (e.g., Janke et al., 1994;ardoya and Meyer, 1996).We describe here a PCR-based approach to completeitochondrial genome sequencing and provide se-

uences of the primers we have used for work on birdsnd other vertebrates. This PCR-based approach al-ows a more rapid determination of complete mtDNAequences than a traditional approach using clonedtDNA. In addition, these primers should make avail-

ble a wider range of mitochondrial genes for smallerequencing studies and, based on comparisons withublished sequences, many of the primers will havetility for other vertebrates. Covering the entire mito-hondrial genome with a focus on birds, this paperugments previous compilations of PCR primers forroader taxonomic categories (e.g., Palumbi, 1996; Si-on et al., 1994) and more specialized primer sets

eported in many studies of birds (e.g., Cooper, 1994;dwards et al., 1991; Edwards, 1993; Lee et al., 1997;uinn and Wilson, 1993; Tarr, 1995) and other reptiles

e.g., Kumazawa and Nishida, 1993; Quinn and Mind-ll, 1996).Targeting highly conserved regions in published ver-

ebrate mtDNA sequences and new sequences that weenerated, we designed pairs of PCR primers thatould amplify the entire mitochondrial genome in

ragments of 3–5 kb (Fig. 1). We used hot-start, XL-PCRith rTth DNA polymerase (Perkin–Elmer) to amplify

hese large fragments: 50-µl reactions included 1.25M Mg(OAc)2, 0.2 mM each dNTP, 0.4 µM each primer,unit rTth DNA polymerase-XL, and 100 ng of totalNA prepared with a QIAamp Tissue Kit (Qiagen).nnealing temperatures were from 55 to 60°C. PCRroducts were gel-purified in 1.5% low-melt agarose,xcised from the gel, and recovered with a Gel Extrac-ion Kit (Qiagen). Double-stranded PCR products were

equenced directly in cycle sequencing reactions using

1055-7903/99 $30.00Copyright r 1999 by Academic PressAll rights of reproduction in any form reserved.

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aq DNA Polymerase FS (Applied Biosystems). Reac-ion products were run on an Applied Biosystems 377utomated DNA sequencer. Working with several taxaimultaneously, we ‘‘walked’’ in on both strands of thesearge fragments and designed additional primers atbout 500-base intervals such that we now have aomplete set of primers for both strands of the avianitochondrial genome. We sequenced the entire mtDNA

f five birds (peregrine falcon, Falco peregrinus; red-ead, Aythya americana; village indigobird, Vidua cha-ybeata; grey-headed broadbill, Smithornis sharpei;nd greater rhea, Rhea americana), and a paintedurtle (Chrysemys picta), and 65 and 95% of the ge-ome, respectively, of an ostrich (Struthio camellus)nd an American alligator (Alligator mississippiensis).omplete mtDNA sequences for the latter three speciesere recently published (Harlid et al., 1997, 1998;anke and Arnason, 1997).A number of standard considerations went into our

rimer design process (e.g., Palumbi, 1996). We tar-eted primers to conserved portions of ribosomal orransfer RNAs or to strings of conserved, twofoldegenerate amino acids in protein coding regions.rimer sequences were evaluated with MacVector (Ko-ak) to ensure adequate annealing temperature andase composition (generally .50% Gs plus Cs) and tovoid secondary structure and self annealing. We de-igned most primers with relatively high annealing

TABLE 1

Approximate Representation of Mitochondrial Genesamong 2023 Avian mtDNA Sequences in GenBank (as

of 22 April 1998)

Gene No. Percentage of total

ytochrome b 1227 612S rDNA 268 13ontrol region (D-loop) 217 11ADH dehydrogenase subunit 2 (ND2) 82 4ytochrome oxidases (mostly COI) 72 46S rDNA 61 3ADH dehydrogenase subunit 6 (ND6) 35 2thers 60 3

FIG. 2. Primers compared to homologous sequences of a varietytrand and position of the 38 base in the published chicken sequenceevised primer) and lowercase letters indicate sites accommodated by, CGT; D, AGT; H, ACT; K, GT; M, AC; N, ACGT; R, AG; S, CG; V, ACG

ine separates birds from other vertebrates. Where two primer sequenecond is a revised version (with changes underlined) that we would ul., 1991; (b) Kocher et al., 1989; (c) Miranda et al., 1997; (d) Sorenequence is followed by homologous sequences for the taxa sequencedtruthio camellus; FAPE, Falco peregrinus; VICH, Vidua chalybeatahrysemys picta; GenBank Accession Nos. AF069422–AF069431) plu

apiens (HOSA, J01415; Anderson et al., 1981), Mus musculus (MUMt al., 1994), Ornithorhynchus anatinus (ORAN, X83427; Janke et alacustre (CRLA, M91245; Tzeng et al., 1992), and Cyprinius carpiolligator are supplemented by published sequences for these taxa

espectively).

emperatures (55–60°C or higher) to facilitate XL-PCR.rimers included a G or C on the 38 end wheneverossible to strengthen primer-template annealing athis important position. We also considered the relativetrengths of unconventional base pairings (see Kwok etl., 1990) and, for example, used G rather than A in therimer when the template was variably C or T and usedrather than C when the template was variably A or G.inally, we incorporated from one to six degenerateositions in many primers, thereby accommodatinguch of the variation in mtDNA sequence among taxa.e have not explored the use of inosine in lieu of

egenerate sites (e.g., Christopherson et al., 1997).One potential pitfall of ‘‘universal’’ primers is the

referential amplification of nuclear sequences of mito-

FIG. 1. Schematic diagram of avian mtDNA showing the loca-ions of light-strand primers listed in Fig. 2. Heavy-strand primersre shown as shorter lines between light-strand primers, but are notabeled. See Fig. 2 for primer sequences. Large fragments typicallymplified in our PCR-based approach to complete mtDNA sequencingre shown outside the mtDNA map. The small subunit (12S) rRNAene was amplified with L1263 and H2294. The gene order shown ishat of Gallus gallus (Desjardins and Morais, 1990), which is sharedy many but not all birds (Mindell et al., 1998).

avian and other vertebrate mtDNAs. L and H numbers refer to thesjardins and Morais, 1990). Dots indicate match with the primer (orenerate positions in primer. Codes for degenerate sites are as follows:, AT; Y, CT. Uppercase letters indicate mismatches with the primer. Aare listed, the first sequence is the primer used in our study and the

n future work. Previously published primers are noted: (a) Mindell etand Quinn, 1998; (e) A. Cooper (in Edwards, 1993). Each primer

ur study (AYAM, Aythya americana; RHAM, Rhea americana; STCA,MSH, Smithornis sharpei; ALMI, Alligator mississippiensis; CHPI,allus gallus (GAGA, X52392; Desjardins and Morais, 1990), Homo01420; Bibb et al., 1981), Didelphis virginiana (DIVI, Z29573; Janke

996), Xenopus laevis (XELA, M10217; Roe et al., 1985), CrossostomaYCA, X61010; Chang et al., 1994). Our sequences for Struthio and12025; Harlid et al., 1997 and Y13113; Janke and Arnason, 1997,

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107PRIMERS FOR MITOCHONDRIAL DNA SEQUENCING

108 SORENSON ET AL.

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109PRIMERS FOR MITOCHONDRIAL DNA SEQUENCING

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110 SORENSON ET AL.

FIG. 2—

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111PRIMERS FOR MITOCHONDRIAL DNA SEQUENCING

FIG. 2—

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112 SORENSON ET AL.

hondrial origin (Zhang and Hewitt, 1997). Becausehey evolve more slowly, these nuclear copies may beore similar to ancestral mtDNA sequences than is the

ontemporary mtDNA (Sorenson and Fleischer, 1996;ischler et al., 1995) and ‘‘consensus’’ primers tend topproximate ancestral sequences. We suggest thatrimers with appropriate degenerate sites are lessikely to preferentially amplify nuclear pseudogenesecause they accommodate likely differences betweenuclear and mtDNA sequences (e.g., 3rd positionshanges in the mtDNAcopy). The high ratio of mitochon-rial to nuclear genome copies in most tissue samples ishen allowed to determine the PCR product. Initialmplification of long mitochondrial fragments and/orse of purified mtDNA will also reduce the risk ofnintended amplification of nuclear pseudogenes (So-enson and Quinn, 1998).Figure 2 compares primer sequences used in our

tudy with a variety of vertebrate mtDNA sequences.e show only primers that we consider useful for most

irds, with the exception of some control region primershat probably have more limited utility. A number of

FIG. 2—Continued

dditional taxon-specific primers, most in ND5, ND6, i

nd the control region, were required to complete eachenome, particularly for the turtle and alligator samplesSorenson et al., in prep). We also list previouslyublished primers that we used and our suggestions forodifications to some of these. Note that the relative

ositions of a given pair of primers and the expectedroduct size may differ in taxa with gene orders differ-nt from that of chicken (see Mindell et al., 1998). PCRrimers can be effective with up to 4 or 5 internalismatches (Christopherson et al., 1997), but are very

ensitive to mismatches in the first few bases of the 38nd (Kwok et al., 1990). A codon rearrangement nearhe beginning of ND6 (Moum et al., 1994) makesrimers in this location (L16206, L16225, H16191)nsuitable for mammals. We have used all of the listedrimers in standard PCR with annealing temperaturesf 50–55°C and with 50-µl reactions including 2.5 mMgCl2, 0.25 mM each dNTP, 0.5 µM each primer, 1.25

nits Taq DNA polymerase, and 100 ng of genomicNA.With these primers in hand, there are two ap-

roaches to sequencing large mtDNA fragments. First,ong amplification products can be sequenced directlyith a number of different internal primers. Using thispproach with six additional birds (Dendrocygna ar-uata, Scolopax minor, Otus asio, Mycteria americana,uteo jamaicensis, Sayornis phoebe; Mindell et al.,npublished data), we sequenced a 4.4-kb PCR product

L2258–H6681, see Fig. 1) with the 2 PCR primers and4 internal primers. On average, 14 primers workedell for each taxon, with different primers failing forifferent taxa. A second approach, which yielded moreonsistent results, was to use the initial, long PCRroduct as a template for the amplification of 500- to00-bp fragments and then to sequence each of theseroducts with the PCR primers used in the secondmplification. This second amplification provides for aerfect match between primer and template in theequencing reaction. We also found that shorter frag-ents that would not amplify from total DNA prepara-

ions for a particular taxon often could be amplifiedrom longer products that included the target sequence.CR was apparently less sensitive to primer mis-atches than were the cycle-sequencing reactions.Recent studies reporting complete mtDNA sequences

ave emphasized the use of ‘‘natural’’ clones of mtDNAather than PCR-amplified DNA (e.g., Harlid et al.,997, 1998; Xu and Arnason, 1996), implying that aCR-based approach is in some way inferior. We ex-lored the question of accuracy by comparing ourstrich, rhea, and alligator sequences with publishedequences for these taxa. Our ostrich sequence (10714p) is 99.75% identical (n 5 27 differences in 19 sepa-ate locations) to the sequence reported by Harlid et al.1997), our rhea sequence (16704 bp) is 99.67% identi-al (n 5 55 differences in 44 separate locations, exclud-

ng a 12-base indel in a repeat region) to the sequence

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113PRIMERS FOR MITOCHONDRIAL DNA SEQUENCING

eported by Harlid et al. (1998), and our alligatorequence (15899 bp) is 99.80% identical (n 5 32 differ-nces in 21 separate locations) to the sequence reportedy Janke and Arnason (1997). Because samples fromifferent individual animals were used in each study,any differences (e.g., those representing third posi-

ion transitions) can be attributed to intraspecific varia-ion. At other positions, however, the previously pub-ished sequences include unusual amino acideplacements that are inconsistent with patterns ofequence conservation among diverse avian taxa (e.g.,ig. 3), as well as insertions, deletions, or transversionshat are inconsistent with conserved blocks of ribo-omal or transfer RNAs. In contrast, our sequences inhese conserved locations are almost always consistentith sequences of other vertebrates. Such comparisons

uggest that errors in the previously published se-uences account for at least 4 of 19, 11 of 44, and 8 of 21ocations that differ between the two ostrich, two rhea,nd two alligator sequences, respectively. By the sametandards, our alligator sequence includes one suspectocation in the ND3 gene, in which the two alligatorequences are temporarily offset by one base, resultingn five consecutive mismatches. Our sequence in thisocation is consistent, however, with the ND3 aminocid sequence of another crocodilian, Crocodylus poro-us (Mindell et al., 1998), although both differ fromther vertebrates for two consecutive positions.Based on these comparisons, we conclude that se-

FIG. 3. Example of a discrepancy between the ostrich mtDNAequence obtained in this study and a previously published sequence.NA and amino acid sequences are shown for a small portion of theOI gene. Asterisks mark positions otherwise conserved among theine ‘‘reptilian’’ taxa. STCA1, this study; STCA2, Harlid et al. (1997).ther taxa as in Fig. 2. STCA2 appears to be offset by one base due to

he inclusion of an extra G, which is then compensated by a missing Geven bases downstream. If translated, STCA2 would include anmino acid substitution in the fourth codon at a site conserved amongirds and most other vertebrates.

uences derived from our PCR-based approach are

ore accurate than previously published sequencessee also Waddell et al., in press). While it is importanto know whether there are any inherent differences inata quality between cloning-based and PCR-basedpproaches to mtDNA sequencing, the above compari-ons do not represent a controlled experiment. Weuspect that errors arising during the interpretationnd transcribing of raw sequence data are more likelyhan errors associated with some systematic differencen replication error associated with cloning versusCR.

ACKNOWLEDGMENTS

We thank Christine E. Thacker and Laura J. Howard for labora-ory assistance. NSF Grant BSR-9496343 to D.P.M. and the Univer-ity of Michigan Museum of Zoology Bird Division Swales and Fargounds supported this research. M.D.S. was supported by NSF GrantBN-9412399 to Robert B. Payne.

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