DNA Isolation, Amplification, and Sequencing

Total genomic DNA extraction of references specimen was performed according to previously described procedures used for Clupeidae studies: a phenol/chloroform/isoamyl alcohol method (PCI) (13,24) and a Chelex method (Bio-Rad, Hercules, CA) (25).

For commercial processed products, oil and lipids were removed from sample tissue by soaking them in water before the DNA extraction. Subsequently, they were washed using 70 % ethanol followed by a second washing step with distilled water. DNA of “defatted” samples was isolated following the same procedures as described above.

PCR amplifications were carried out using Hybaid PCR Express (Hybaid, Ashford, U.K.) and Bio-Rad MyCycler™ (Bio-Rad, Hercules, U.S.A.). The reactions were set up in volumes of 50 µL (references species) or 100 µL (processed products) containing PCR buffer [75 mM Tris-HCl, pH 9.0; 50 mM KCI; 20 mM (NH4)2SO4, 2 mM MgCI2]; 400 µM dNTP mix; 0.2 µM of each primer; 2.5 units of UptiTherm DNA polymerase (Uptima-Interchim, Montluçon, France); and 0.5-1 µL of template DNA (PCI extracts) or 1-3 µL of DNA solution (Chelex® supernatant).

Cycling conditions were:

  • For the 16S gene: 94°C for 5 min, 35 cycles (94°C for 30 s, 55°C (long and short DNA fragments) for 30 s, 72°C for 60 s), followed by a final extension for 5 min at 72°C;
  • For the COI gene: 94°C for 180 s, 35 cycles (94°C for 30 s, 48°C (long DNA fragment) or 48°C to 55°C (short DNA fragment depending of the primers) for 30 s, 72°C for 30 s), followed by a final extension for 5 min at 72 °C;
  • For the cytb gene: 95°C for 5 min, 35 cycles (94°C for 30 s, 50°C for 45 s, 72°C for 60 s), followed by a final extension for 5 min at 72°C (long DNA fragment) and 94°C for 180 s, 35 cycles (94°C for 30 s, 55°C for 30 s, 72°C for 45 s), followed by a final extension for 5 min at 72°C (short DNA fragment).

For optimal results in some cases the cycling conditions were appropriately adapted in terms of cycling number (between 30 and 40) and annealing temperature.

The DNA amplification was controlled on 1.5 to 3 % agarose gels (Agarose HR, Uptima-Interchim), using TAE buffer (2 mM EDTA, 40 mM Tris acetate, pH 8.5). Ethidium bromide was used for band visualization via ultraviolet transillumination (Image Master VDSCL, Amersham Pharmacia Biotech, Freiburg, Germany). The size of expected PCR products was estimated using the GeneRuler™ 100bp DNA ladder Plus and the GeneRuler™ DNA ladder mix (MBI Fermentas, Lithuania). For sequencing reactions the DNA template was quantified using the MassRuler™ DNA ladder, low range (MBI Fermentas, Lithuania) on agarose gels.

For the reference species three sets of primer pairs (table 2) were used in the PCR amplifications to obtain 600/700 bp DNA fragments of 16S rRNA (Miya and Nishida (28)), COI (Folmer et al. (29)), and cytb (Sevilla et al. (30)).

Based on the DNA sequences determined for these DNA fragments we designed three primer sets (table 2, short DNA fragment 200/300 bp) for the amplification of short species-distinctive fragments of the COI, 16S rRNA and cytb genes. For each gene as primer anchor regions two conserved zones flanking the diagnostic target region of the examined species, especially the five main Engraulis species, were identified.



Primer sequence 5’-3’






Bautista (24)




Bautista (24)

Cytb (short DNA fragment)



This study




This study



MIY L2510

Miya – Nishida (22)



MIY H3059

Miya – Nishida (22)

16S (short DNA fragment)



This study




This study




Folmer et al (23)




Folmer et al (23)

COI (short DNA fragment)



This study




This study

Table 1. Primer Sequences for PCR amplifications of long and short DNA fragments of the 16S, COI and Cytb genes gene, 16S gene, COI gene and a short fragment of COI.


Cleanup and Sequencing of PCR Products

Prior to sequencing, double-stranded PCR products were purified by filtration through a Qiagen QIAquick column according to the manufacturer’s protocol. PCR fragments were used for direct cycle sequencing with the Dye Terminator Cycle Sequencing sequencing kit (Beckman, Villepinte, France).
Sequencing analysis was performed with a Beckman Coulter CEQ 8000 DNA sequencer (Beckman, Villepinte, France) in both directions with the primers used for PCR amplification.

Sequence Alignment, Genetic Distances and Phylogenetic Analyses

The DNA sequences were edited with BioEdit software (31). Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 3.1 (Kumar, Tamura, Nei (2004) (32)). Nucleotide divergences were computed using Tamura-Nei model (33), which takes substitutional rate biases and the inequality of base frequencies into account. Phylogenetic trees were constructed using the neighbor-joining method (34) and the robustness of topology nodes was tested by the bootstrap method with 1500 iterations.