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236 Endang Species Res 27: 233–241, 2015
to 3 products were sequenced in both forward and polymorphic site, the remaining amplicons allowing
reverse directions to ensure the veracity of results. adequate haplotypic assignment (Pardini et al. 2001).
Sequence chromatograms were screened independ- Two additional primers were designed to target poly-
ently by eye and verified by colleagues to ensure morphisms diagnostic of potential Atlantic haplo-
accuracy of base calling. types as distinct from those of Mediterranean or
Pacific origin (Gubili et al. 2011). Of these, only one
(D-loop7) was successfully used in this analysis,
MtDNA analyses yielding a 206 bp product.
Ten nanograms of genomic DNA were used for
Five pairs of primers were designed using Primer 20 µl PCRs (polymerase chain reactions) containing
Premier 5.0 (www.PremierBiosoft.com) to amplify 1× NH4 buffer, 200 µM of each dNTP, MgCl2 (1.5–
independently 5 overlapping fragments, from 135 to 2.5 mM; Table 2), 0.3 µM of each primer, and 1.0 U of
286 bp, of the D-loop sequence (Table 2, Fig. 1). AmpliTaq Gold™ DNA polymerase (Applied Bio-
However, due to A+T-rich regions of the mtDNA, systems) on a Biometra T-Gradient thermal cycler.
there are necessarily overlaps with 3 pairs: D- Amplification conditions consisted of initial denatu-
loop1Reverse with D-loop2Forward (9 bases), D- ration for 5 min at 94°C, followed by 40 cycles of 30 s
loop3Reverse with D-loop4Forward (11 bases), and at 94°C; 30 s annealing (temperature dependent on
D-loop4Reverse with D-loop5Forward (14 bases). the primers used; Table 2), 30 s at 72°C; and a final
Nevertheless, each overlap contained only a single extension step of 10 min at 72°C. The same quantity
of genomic DNA from historical
Table 2. Carcharodon carcharias. Primer sequences and PCR (polymerase chain material was used for an initial 20 µl
reaction) conditions for amplification of overlapping fragments of the white PCR using the primers GWSF6
shark mtDNA D-loop (5’ TTG GCT CCC AAA GCC AAG
ATT CT 3’) and PheCaCaH (5’ CTA
Primer Primer sequence (5’−3’) Annealing Amplicon MgCl2 CTT AGC ATC TTC AGT GCC 3’)
temp. (°C) size (bp) (mM) (Gubili 2009) to achieve partial am-
D-loop1 ACA CGC ACG TAT ATT GCT AAC TG 54 135 2.5 plification of the full mitochondrial
CCA AAA CTG AAA GGG ATA GAG AG 54 135 2.5 D-loop. The resulting PCR products,
including all negative controls, were
D-loop2 ATT ATG GCG TCA ATC TCT CTA TC diluted 1/10, of which 1 to 3 µl was
GAG GCT CAT CTG GGA CAC TAA G
D-loop3 TAG AAG AGT GTC GAG GGG AGT AC 54 286 2.5 used for subsequent semi-nested
AAT CCT CAT CAA CTG AAC AAA CC PCRs of 40 µl volume, for all 6 over-
D-loop4 TAA ATG TCA GGT TTG TTC AGT TG 48 228 1.5 lapping primer sets. Positive controls
ATC CCC ATT CAT CTA CTT ACA GC were not used, as other DNA proto-
D-loop5 AAT GAA ATT GCT GTA AGT AGA TG 48 245 2.5 cols for historical studies deem them
CTG AAT GCT GTC AAA ACA TG 206 2.5 to be of little value (Fulton & Stiller
2012). All amplicons were assayed
D-loop7 CGT ATC CAT TAT GGC GTC AAT CTC T 60–63 on 2% agarose gels, purified using
GCG TCA AGA TTT ATT TTC CAC
the QIAGEN QIAquick PCR purifi-
cation kit following the manufac-
turer’s instructions, and commercially
sequenced.
To confirm the suitability of teeth as
a source of DNA, sequences obtained
from South African tooth samples
were aligned to those generated from
fin tissues of the same individuals, us-
ing ProSeq 3.2 (Filatov 2002). To de-
Fig. 1. Carcharodon carcharias. Schematic of partial mitochondrial D-loop termine their similarity to other avail-
sequence. Black bars show primer positions of Amplicons 1 to 5 and 7 of the able haplotypes, sequences derived
D-loop (DL). White box along the partial sequence represents 749 base pairs of from historical Mediterranean sam-
the sequence used in this analysis. Start and stop positions are provided for each ples were aligned to white shark se-
primer amplicon and sequence used, and correspond to regions of the full mito- quences available on GenBank, in-
chondrial DNA (mtDNA)
