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

Keywords: 
 
polymerase chain reaction; 
molecular evolution; 
DNA amplification; 
restriction fragment length polymorphisms

for 1 min, and 72 "C for 1 min, and a final extension at 72°C for 5 min. Amplification products were purified from unincorporated primers using the ' Promega Wizard PCR Preps DNA Purification System'. Heavy and light strands of amplified fragments were cycle-sequenced from one specimen per species at the Molecular Genetics Facility, University of Georgia, using the fluorescent-dye chain-terminator method (Applied Biosystems automatic sequenator). Sequences were aligned with the computer program Sequence Navigator (Applied Biosystems) and five regions (ranging from 32 to 41 bp in length) of 100% sequence similarity across the armadillo species were noted. From the two longest of these regions (36 and 41 bp), 32-bp primers were designed with assistance from the program Oligo (National Biosciences, Inc.): Dnovl6Sfor (5'-AATAGGGTTTACGACC-Dnoul6S-rev (S-TGATTATGCTACCTTTG-These primers then were employed in a three-step amplification using a Hybaid Thermal Cycler and the Expand" Long Template PCR system from Boehringer-Mannheim (B-M). This system jointly employs two polymerases: a nonproofreading Taq that is the main polymerase in the reaction, and a proofreading Pwo at lower concentration (see the B-M manual for further details). Optimized conditions, achieved according to the manufacturer's recommendations, were as follows, carried out in 50-pL reaction volumes: 1 x buffer 3 [whose 10 x stock contains 22.5 m~ MgCI,, 500 m~ Tris-HC1 pH 9.2, 160 m M (NH,),SO, 20% v/vDMSO, 1% v / v Tween 20],2.5 units enzyme mix, 100 ng of genomic DNA or 50-100 ng of CsCl gradient-purified mtDNA, 350 p~ of each dNTP, and 25 pmol of each long-PCR primer. Cycling parameters consisted of initial denaturation at 94 "C for 2 min; 10 cycles at 92 "C for 30 s, annealing at 64 "C for 30 s, and TCGATG'ITGG ATCAGG-3' ); CACGGTCAGGGTACC-3' ). elongation at 68 "C for 12 min; another 25 such cycles with 20 additional seconds for the elongation step per cycle; and a final extension period of 7 min at 68 "C.
Each initial check for proper amplification of fulllength (c. 16.7 kb) mtDNA involved electrophoresis of 3 pL of the reaction mixture along side a 1-kb ladder standard (Bethesda Research Laboratories) through a 1% agarose gel (1 x TBE), followed by EtBr visualization.
From the dosed-circular mtDNAs (c. 500 ng), as well as from 3 pL of the linear full-length mtDNAs amplified by long-PCR, restriction digests were performed according to the manufacturer's recommendations using DraI, EcoRI, EcoRV, HindII, HindIII, HpaII, and TnqI. MtDNA fragments from the CsC1-based preparations were radioactively end-labelled with %tagged nucleotides before electrophoresis (Lansman et al. 1981). Restricted mtDNA fragments from the long-PCR were revealed by EtBr staining following electrophoresis. In all cases, mtDNA fragments were separated through 1.5% agarose gels and sized against 1-kb ladders..
The long-PCR primers successfully amplified fulllength linear mtDNA from all armadillo species examined.
The Dnovl6S-fir and Dnml6S-rev primers are separated by 366 bp in Dnsypus mtDNA, so by 'full-length' amplification we mean that about 98% of the mtDNA molecule was copied. The initial gauge of success was the appearance of single DNA bands of appropriate size (c. 16-17 kb) in EtBr-stained agarose gels whose lanes had been loaded with reaction mixtures resulting from amplification by the long-PCR primers described above. Lanes with negative controls failed to show these bands. Successful amplification of full-length mtDNA was confirmed by agreement of restriction digestion profiles between the mtDNAs isolated from long-PCR and those from conventional CsCl gradient purifications (Figs 1, 2).
The right-hand lanes in Figs l@) and 2@) indicate that the Dna, long-PCR primers also amplified full-length mtDNA suitable for restriction digestion from total genomic preparations from each of the three other armadillo genera examined. Furthermore, 75% of the 20 long-PCR attempts succeeded when the starting template involved genomic DNA preparations from the ethanolpreserved D q u s ear clips. Preliminary attempts to amphfy full-length mtDNA from other vertebrates using the long-PCR armadillo primers met with mixed success. Species yielding an amplification product of expected full-length mtDNA size (c. 16-17 kb) included the fox squirrel Sciurus n i p , snow goose Chen (or Anser) cuerulesctns, chuckwalla Snuromalus obesus, desert iguana Dipsosaurus dorsalis, western garter snake Tkumnophis ekgans, spring salamander Gyn'nophilus porpkyriticus, rainbow trout Oncorhynchus mykiss, and Atlantic salmon Salmo salar. However, each of these species also displayed smaller fragments in the gels per- Fig. 1 HpaII digests of armadillo mtDNA. (a): autoradiograph based on end-labelling of closed-cimrlar mtDNA as isolated by conventional CsCl gradient centrifugation from 15 specimens of Dasvpus novnncirlctirs. The seventh lane from the right is a I-kb ladder size standard. @) EtBr-stained gel in which mtDNAs had been amplified by long-PCR. The lefhnost lane is a 1-kb molplar size standard, lanes 2-13 are Dasypus novemcinctus, lanes 14-17 Chuetophractus sp., lane 18 Tolypeutes matacus, and lanes 19-20 Zoedyus yichiy. For Dnsypus, note the appearance in Figs l(a) and 1@) of the same seven mtDNA fragments ranging in size from about 0.7 to 1.9 kb. This outcome also suggests that one of the HpnU restriction sites is close to the PCR priming sites, an inference confirmed by direct examination of the 165 rRNA gene sequence where a Hpn restriction site occurs within the 366 bp region excluded from long-PCR amplification. Digestion profiles p i e d u d by three other restriction enzymes (DraI, Hindu and TqI) similarly appeared identical in assays of both long-PCR and CsCIisolated mtDNA. Fig. 1 and  the text). For this enzyme, as well as for EcoRV and Hindu, digestion profiles from the long-FCR products were different from those of conventional digests in ways reflective of the fact that long-PCR mtDNA products are linear as opposed to circular. For example, conventional EcoRI digestions of closedcircular mtDNA in Dusypus novrmcinctus revealed two fragments of sizes 3.4 and c. 13 kb (Fig. Za), whereas digestions of the linear long-PCR products revealed three fragments of sizes 3.4, and c. 6 and 7 kb (Fig. 2b). In this case, the priming sites for long-PCR occur near the middle of the 13-kb fragment obtained in the conventional digestion Such differences in digestion profiles also further confirm that the latter reflect PCR-amplified mtDNA product as opposed to mere presence of CsCl gradient-purified mtDNA.

Fig. 2 EcoRI digests of armadillo mtDNA (see legend to
haps indicative of occasional mis-priming or lack of primer specificity for the 16s rRNA gene. Species failing to yield a full-size mtDNA amplification product in our preliminary assays were the sooty tern Sterna fuscata, sharptailed sparrow Ainrnodramus caudacutus, green turtle Chelonia mydas, and spotted bass Micropterus punctulatus. Finally, a search of nucleotide sequences in GenBank was conducted to identify targets with 100% identity to the Dnou26S-for and Dnov26S-rm long-PCR primer sequences. Available 16s rRNA gene sequences matched both primers perfectly for the three-toed sloth Bradypus variegatus, two-toed sloth Choloepus didactylus, extinct ground sloth Mylodon dnnuinii, another armadillo species Cabassous rrnicinctns, and proboscis monkey Nasalis larvatus. Sloths (and anteaters) also belong to Edentata and are thought to be close relatives of armadillos (Hoss et al. 1996). Another 105 species had sequences that matched either Dnarl6S-for or l h o v 2 6~-r a r .
These results, together with the empirical findings described above, suggest that the armadillo-derived long-PCR primers may amplify fulllength mtDNA from some other vertebrates also. However, further evaluation will require species-specific experimentation with varied PCR conditions.
Following early attempts to amplify lengthy DNA fragments in vitro (e.g. Kainz et al. 1992;Ponce & Micol 1992), 'long-PCR methods were refined by Barnes (1994) and Cheng et al. (1994a) who demonstrated high fidelity amplification of phage h templates over 35 kb. Long-PCR relies on stringent annealing of long primers (permitting high annealing temperatures and thus a reduced risk of false priming), polymerase proofreading activity, and short high-temperature denaturations coupled with exacting temperatures of elongation.
In population and evolutionary genetics, several advantages can be envisioned for the assay of whole mtDNA by long-PCR (a) laborious and time-consuming steps of physical mtDNA isolation by conventional methods are circumvented; @) the isolations do not require large amounts of starting tissue, nor inevitable sacrifice of the organism; and (c) mtDNA amplification product should be unlimited, thus permitting fast gel screening by methods (e.g. EtBr staining) that avoid sensitive and expensive detection techniques. Additional advantages are that: (a) some population survey methods (e.g. by restriction enzymes that cut at five-and six-bp recognition sites) are better suited for full-length animal mtDNA than for short gene sequences within it (because of the scorable number of fragments typically produced); @) extensive data from traditional whole-mtDNA surveys are available for many species for comparison; and (c) the priming site in long-PCR provides a consistent (across enzymes) anchor for restriction site mapping.
Potential disadvantages of long-PCR include those that apply to any PCR-based method: (a) the effort involved in developing suitable primers and assay conditions for the species in question; and (b) the danger of amplification from non-target DNA. As applied to animal mtDNA, this latter concern is in one respect diminished in long-PCR as compared with regular PCR applications. One prominent source of nonspecific amplification occurs when PCR primers developed expressly for gene sequences in mitochondria 'inadvertently' amplify paralogous sequences in the nucleus. Nudear transfer of short mtDNA sequences is a rather common and ongoing phenomenon in animals and plants (Blanchard & Schmidt 1995, 19!36), and the resulting mtDNA pseudogenes can create difficulties (as well as novel opportunities) in population and evolutionary analysis (Smith ef nl. 1992;Arctander 1995;Dowling et nl. 1996;Zhang & Hewitt 1996). Such complications are less likely to compromise mtDNA long-PCR because no transfers of full-length mtDNA to the nucleus have yet been reported.
We have demonstrated the technical feasibility of PCR amplification of full-length animal mtDNA. The current results involving armadillos suggest that with further refinement, the long-PCR approach for animal mtDNA may find a variety of applications in population genetic and evolutionary studies.