DNA primers for amplification of mitochondrial cytochrome ...

1 Molecular Marine Biology and Biotechnology(1994) 3(5), 294-299 DNA primers for amplification of mitochondrial cytochrome coxidase subunit I from diverse metazoan invertebrates0. Folmer, M. Black, W. Hoeh,* R. Lutz, andR. Vrijenhoek+Center for Theoretical and Applied Genetics, andInstitute of Marine and Coastal Science, RutgersUniversity, New Brunswick, New Jersey 08903-231 AbstractWe describe "universal" DNA primers for polymer-ase chain reaction (PCR) amplification of a 710-bpfragment of the mitochondrial cytochrome c oxidasesubunit I gene(COI)from11invertebrate phyla:Echinodermata, Mollusca, Annelida, Pogonophora,Arthropoda, Nemertinea, Echiura, Sipuncula, Pla-tyhelminthes, Tardigrada, and Coelenterata, as wellas the putative phylum Vestimentifera. Preliminarycomparisons revealed that theseCOIprimers gener-ate informative sequences for phylogenetic analysesat the species and higher taxonomic purpose of this short communication is to de-scribe "universal" DNA primers for the polymerasechain reaction (PCR) amplification of a 710-bp frag-ment of the mitochondrial cytochrome c oxidasesubunit I gene(COI).

2 This study was motivated bythe recent discoveries of more than 230 new inver-tebrate species, comprising new genera, families,classes, orders, and potentially a new phylum, fromdeep-sea hydrothermal vent and cold-water sulfideor methane seep communities (Tunnicliffe, 1991).Our goal was to develop molecular techniques forphylogenetic studies of these diverse organisms. Wefocused on the mitochondrial cytochrome c oxi-dase subunit I(COI)gene because it appears to beamong the most conservative protein-coding genesin the mitochondrial genome of animals (Brown,1985), which was preferable for the evolutionary*Present address: Department of Biology, Dalhousie Univer-sity,Halifax, Nova Scotia, Canada.+Correspondence should be sent to this 1994 Blackwell Science, depths likely to be found in our quickly became aware of the broad utility oftheseCOIprimers for broader systematic studiesof metazoan invertebrates, including acoelomates,pseudocoelomates, and coelomate protostomes design candidate primers , we compared pub-lished DNA sequences from the following species:blue mussel,Mytilus edulis;fruitfly,Drosophila ya-kuba;honeybee,Apis mellifera;mosquito,Anophe-les gambiae;brine shrimp,Artemia franciscana;nematodes,Ascaris suumandCaenorhabditis ele-gans;sea urchin,Strongylocentrotus purpuratus;carp,Cyprinus carpio;frog,Xenopus laevis;chicken,Gallus gallus;mouse,Mus musculus; cow,Bos taurus;fin whale,Balaenoptera physalus;andhuman,Homo sapiens(Figure1).

3 Several highlyconserved regions of theseCOIgenes were used asthe targets for primer , three coding-strand and six anti-coding-strand primers were tested (Table1)for am-plification efficiency. The following primer pairconsistently amplified a 710-bp fragment ofCOIacross the broadest array of invertebrates:LCO1490: 5'-ggtcaacaaatcataaagatattgg-3'HC02198: 5'-taaacttcagggtgaccaaaaaatca-3'In the code names above, L and H refer to lightand heavy DNA strands, CO refers to cytochromeoxidase, and the numbers (1490 and 2198) refer tothe position of theD. yakuba 5' also present the primers as coding-strand se-quences, along with their inferred amino acids (Fig-ure1).The usefulness of these primers results fromthe high degree of sequence conservation in theirrespective 3' ends across the 15 taxa. The 3' end ofeach primer is on a second-position nucleotide.

4 Allother pairwise primer combinations amplifiedfewer taxa or gave additional nonspecific productsunder less stringent amplification LCO1490 and HC02198 amplified DNAfrom more than 80 invertebrate species from 11 Figure sequences of the LCO1490 andHC02198 primers and inferred amino acid represent identical nucleotides at a given positioncompared withDrosophila yakuba.*Position as listed inGenBank. Accession numbers and primary references forGenBank sequences are as follows:Mytilus edulis,M83761/M83762 (Hoffmann et al., 1992);Drosophila ya-kuba,X03240 (Clary and Wolstenholme, 1985);Apis mel-ifera,M23409 (Crozier et al., 1989);Anopheles gambiae,L20934 (Beard et al., 1993);Artemia franciscana ( , direct submission to GenBank access numberX69067);Strongylocentrotuspurpurat us, X12631 (Jacobset al., 1988);Ascarissuum, X54252, andCaenorhabditiselegans,X54253 (Okimoto et al.)

5 , 1990);Cyprinus carpio,X61010 (Chang and Huang, 1991);Homo sapiens,M12548(Anderson et al., 1981);Mus musculus,V00711 (Bibb etal., 1981);Bos taurus,V00654 (Anderson et al., 1982);Balaenoptera physalus,X61145 (Arnason et al., 1991);Xenopus laevis,X02890 (Roe et al., 1985); andGallusgallus,X52392 (Desjardins and Morais, 1990).phyla (Table2).The PCR products of species fromfive phyla (Mollusca, Annelida, Arthropoda, Vesti-mentifera, and Coelenterata) are illustrated in forHydra,all products resulted froma single PCR amplification . TheHydrasample wasreamplified to provide sufficient product for directsequencing. For several species, initial amplifica-tion produced multiple PCR products. In thesecases, target DNA for sequencing was obtained byraising the annealing temperature, or gel-isolatingthe initial 710-bp fragment and reamplifying verify that the amplified fragment is indeedCOI,we obtained a minimum of200by of se-quence from all species listed in Table2(exceptthose marked with an asterisk).

6 Typically, cycle-Universal COIprimersfor invertebrates295sequencing with these primers produced a readablesequence of at least 651 bp, equivalent to219in-ferred amino acid residues. To demonstrate that theproducts areCOI,we provide four new sequences(in reading frame) from work in progress on deep-sea invertebrates (Figure 3). Comparisons of thesesequences that mostvariation occurs at the third-position analyses of thisCOIfragment from a di-verse array of bivalve mollusks and vestimentiferantube worms suggest that phylogenetic resolution atthe phylum and class level can be obtained frominferred amino sequences. Intermediate-level reso-lution (family to genus) is retained in first- andsecond-position nucleotides. Third-position substi-tutions are saturated at these higher levels, but re-tain informative polymorphisms within at least onebivalve species,Bathymodiolus universalDNA primers ,LCO1490andHCO2198,amplified a 710-bp region of the mito-chondrial cytochrome oxidase subunit I gene froma broad range of metazoan invertebrates.

7 We arepresently using these primers to examine phyloge-netic relations among the following taxa:(1)tubeworms (Vestimentifera) and other protostomeworms (Pogonophora and Annelida);(2)deep-seamarine bivalve mollusks (Mytilidae and Vesicomyi-dae); (3) freshwater bivalve mollusks (Unionidae,Dreissenidae, and Corbiculidae);(4) COI primers tested in this study, pre-sented relative to the coding strand ofDrosophila Folmer, M. Black, W. Hoeh, R. Lutz, and R. VrijenhoekTable representing eleven different phyla for which theLCO1490andHC02198primers amplified andsequenced the 710-bp mitochondrial COI fragment.*Amplified, but not sequenced to datet Jones (1985)Table (Caridae); and (5) parasitic platyhel-minths (Trematoda). We also are investigating theutility of thisCOIfragment for larval identificationsin several of these groups. Independent laboratorieshave verified the utility of the LCO1490 andHCO2198 primers for amplification and sequencingofCOIfrom (1) oysters, generaCrassostrea ( ,Louisiana State University, and M.)

8 Hare, Universityof Georgia) andOstrea(Diarmaid O'Foigel, Univer-sity of South Carolina); (2) scallops, genusPlacopec-ten( , University of Delaware); (3) hardclams, genusMercenaria( 'Foigel); (4) archaeo-gastropod limpets (A. MacArthur, University of Vic-toria); (5) arachnids (A. Tan, University of Hawaii);and (6) marine hydrozoans (S. Karl, University ofSouth Florida).Experimental ProceduresWhole cell DNA was extracted from either freshtissue or tissue frozen at - 80 C immediately aftercollection of a specimen. We used a conventionalhexadecyl-trimethyl-ammonium bromide (CTAB)protocol, modified from Doyle and Dickson (1987).Typically, 1 mm3of tissue was extracted and theL 1 2 3 4 5 6 7 8 LFigure gel of PCR products from seven differ-ent species of invertebrates. All PCR products except lane7 are directly amplified from total DNA extraction. Lane L,Phi-X/HaeIII ladder.

9 Lane 1, blue mussel,Mytilus 2, squid,Loligo 3, polychaeteParalvi-nella 4, oligochaeteTubifex 5, shrimp,Rimicaris 6, tube worm,Riftia 7, reamplification of hydra, 8, negative control PCR reaction with allcomponents except template COI primers for invertebrates297 DNA resuspended in 75 to 150 l(dependent uponthe size of the pelleted DNA) of sterile distilledwater. In our experience, DNA extracted by thisprotocol and stored at - 20 C remains intact for atleast three chain reactionWe typically used 1 lof the DNA extract as tem-plate for a 50- 1 PCR reaction, using 4 units of Taqpolymerase (Promega, Madison, WI) per 50- 1 reaction consisted of 5 ofloxbuffer(provided by the manufacturer), 5 lof MgCl2( , both solutions supplied with the polymer-ase), lof each of the two primer stock solu -tions (10 mol/liter), 5 C, T, A, G nucleotide mix(Boehringer Mannheim, Indianapolis, IN, 2 mol/liter for each nucleotide), and 29 lsterile distilledwater.

10 Reactions were amplified through 35 cyclesat the following parameters: one minute at 95 C,one minute at 40 C, and one and a half minutes at72 C, followed by a final extension step at 72 C forseven minutes. Amplifications were confirmed bystandard submarine gel electrophoresis, using 2%w/v low-melting agarose/TBE gels (NuSieve, FMCBioProducts), stained with ethidium templates could be sequenced from a singleround of amplification . Occasionally, templatesprovided too little product from a single amplifica-tion. In such cases, the first amplification productwas gel-isolated and used as template for a reampli-fication with a higher annealing temperature (50 C,all other parameters being held the same). In allinstances, the PCR product for sequencing was ob-tained by running the entire reaction volume ona 2% low-melting agarose gel, using wide-toothcombs.