NOTE Chan et al.: Identification of Carcharhinus spp, by DNA-based techiniques 



911 



morphism (PCR-RFLP; Martin, 1991), DNA sequencing 

 techniques (Heist and Gold, 1999), isoelectric focusing of 

 muscle proteins (Renon et al., 2001; Smith and Benson, 

 2001) and direct multiplex PCR amplification (Shivji et 

 al., 2002). These techniques use the differences in the se- 

 quences of nucleotide bases within a DNA strand among 

 species. DNA techniques have high sensitivity, are easily 

 reproduced, and allow the development of a unique "DNA 

 fingerprint" for each species (Martin, 1991; Innes et al., 

 1998; Pepperell and Grewe, 1999). It was the aim of this 

 project to initiate a shark DNA database for species iden- 

 tification of pelagic sharks (beginning with species from 

 the genus Carcharhinus) in New South Wales by using 

 PCR-RFLP techniques. 



Materials and methods 



Sharks of the genus Carcharhinus landed by recreational 

 fisheries and caught in NSW beach meshing were identified 

 to species level by using morphometric taxonomic guides 

 and dentition identification (Cliff and Wilson, 1994; Last 

 and Stevens, 1994; Naylor and Marcus, 1994). Positively 

 identified sharks were retained as voucher specimens (see 

 "Acknowledgments" section). A tissue biopsy (5-10 g) from 

 the dorsal region on either side of all voucher specimens 

 and unidentified sharks was taken and stored in 75% etha- 

 nol prior to DNA extraction. Mitochondrial DNA (mtDNA) 

 of specimens from six species of the genus Carcharhinus 

 (C. brachyurus, C. brevipinna, C. falciformis, C. leucas, C. 

 limbatus, and C ohscurus; see Table 1 for sample sizes) was 

 extracted by using a Fastprep DNA Extraction kit (BIOlOl, 

 Integrated Sciences, Sydney, New South Wales) following 

 the manufacturer's instructions. Approximately 200-300 

 mg of tissue was placed in a sterilized 1.5-mL eppendorf 

 tube and after the addition of 1 mL Fastprep lysis buffer, 

 incubated at 56°C for three hours prior to the extraction 

 stage (Chan, 2001). Following the extraction procedure of 

 the Fastprep protocol, quality and quantity of DNA was 

 measured by using a GeneQuant DNA/RNA calculator 

 ( Amersham Biosciences, Sydney, New South Wales). 



The polymerase chain reaction (PCR) was used to am- 

 plify the 1146 nucleotide base-pair (bp) cytochrome b (cyt 

 h) region of the mtDNA (Martin and Palumbi, 1993; Kita- 

 mura et al., 1996). For each 50 pL PCR reaction, 100-200 

 ng of template mtDNA was used, 1.5 niM MgCU, IX PCR 

 buffer, 2 mM of dNTP, 5 mM of each external primer 

 (5'-TGACTTGAARAACCAYCGTTG-3' and 3'-CTCCAG- 

 TCTTCGRCTTACAAG-5') and two units of DyNAzyme 

 EXT DNA polymerase (Finnzymes, GeneWorks, Adelaide, 

 South Australia) were added to a sterilized 200 pL PCR 

 tube. The PCR was undertaken in a MJR MiniCycler (MJR, 

 GeneWorks) with a heated bonnet on a cycle of 94°C for 

 three minutes, followed by 35 cycles of 94°C denaturing 

 for 45 seconds, 48°C annealing for 30 seconds, 72°C exten- 

 sion for 90 seconds, and a final 10-minute extension time 

 at 72°C (Chan, 2001). 



To determine if the cyt 6 region was successfully ampli- 

 fied, 10 pL of PCR product was added to 2 pL loading dye 

 (25% bromophenol blue, 40% sucrose in distilled water) and 



loaded into wells of a 1.5% w/v agarose gel submerged in 

 0.5X TBE (Tris-borate-EDTA, pH 8) buffer, with 1 pg of a 

 100-bp DNA ladder (Sigma-Aldrich, Sydney, New South 

 Wales) added to 5 pL distilled water added to each side- 

 end well. The gel was subject to electrophoresis at 125 V 

 for 45-60 minutes and then stained in ethidium bromide 

 for 10 minutes, de-stained in fresh distilled water for 20 

 minutes prior to illumination under ultraviolet (UV) light 

 to determine the success and yield of the amplification. 



To obtain species-specific profiles, restriction fragment 

 length polymorphism (RFLP) was used on the entire 1146 

 bp cyt b fragment (Martin, 1991; Chan, 2001). The suc- 

 cessful amplified reaction products had the primers, taq 

 polymerase, and buffer chemicals removed by using a 

 BRESAspin PCR purification kit (GeneWorks). For each 

 RFLP reaction, 30 pL of purified PCR-amplified cyt b 

 mtDNA (300-1000 ng; 30 pL of distilled water was used 

 for control reactions), 5 pL of lOX buffer, one unit of a re- 

 striction enzyme (Alu l,Hae III, Ps? I, Taq I,andXAo I) and 

 distilled water up to 50 pL volume was added to a steril- 

 ized 200 pL PCR reaction tube and incubated at 37°C for 

 2 hours in the MJR minicycler (with heated bonnet), with 

 the exception of Taq I (incubated at 65°C for two hours in 

 a water bath). After the allotted digestion time, 10 pL of 

 loading dye was added to each tube prior to loading into a 

 1.5% w/v agarose gel submerged in 0.5XTBE buffer In both 

 end wells, 5 pL of distilled water -i- 1 pg of a 100 bp DNA 

 ladder was added. The gel was subject to electrophoresis 

 at 125 V for 60-90 minutes, stained in ethidium bromide 

 for 10 minutes, and destained in fresh distilled water for 

 20 minutes prior to illumination under UV light. Enzyme- 

 digested DNA fragments >100 bp were then "scored" to the 

 nearest 25 bp based upon migration of the DNA fragment 

 (the smaller the fragment, the faster the migration) and 

 recorded for each enzyme and sample (Martin, 1991) by 

 using the 100-bp DNA ladder as a standard measuring 

 guide for size estimation. 



Results and discussion 



PCR-RFLP profiles were successfully developed for six spe- 

 cies of the genus Carcharhinus; distinct and discrete pat- 

 terns were observed for each species with five restriction 

 enzymes (Table 1, and Chan, 2001). The only intraspecific 

 polymorphism observed was for two specimens of C. bre- 

 vipinna with the Xho I restriction enzyme. Increasing the 

 sample size of all species may identify more intraspecific 

 polymorphisms. Because of the relatively small sample 

 sizes, no statistical analyses were undertaken. Other 

 restriction enzymes were tested (Chan, 2001), and with 

 the possible inclusion of other species from the genus Car- 

 charhinus into this database in the future, these restriction 

 enzymes may be required in order to discern the additional 

 species. Because some of the fragment sizes were rounded 

 to the nearest 25 bp, the sum of the fragments for a restric- 

 tion enzyme of a species may be more than 1146 bp, the 

 size of the C3rt b uncleaved region for sharks (Martin and 

 Palumbi, 1993). Fragments <100 bp were not recorded 

 because the DNA ladder had a lower limit of 100 bp. 



