54 



Fishery Bulletin 97(1), 1999 



ment differences in polymerase chain reaction (PCR) 

 amplified DNA might provide a rapid and inexpen- 

 sive means of identifying carcasses and fins. We de- 

 velop this technique as a means of identifying the 

 eleven most frequently landed carcharhiniform 

 sharks in the U.S. east coast longline fishery. 



Materials and methods 



Tissues (heart, white muscle, or fin ) from nine spe- 

 cies of carcharhinid sharks and two species of 

 sphyrnid sharks were obtained from commercial fish- 

 ermen, sport tournaments, and research longlining 

 cruises (Table 1). Tissues were either frozen in the 

 field and stored at -80°C or preserved immediately 

 in lOx Longmire's lysis buffer (0.1 M tris, 0.1 M 

 NagEDTA, 0.01 M NaCl, 0.5% SDS, pH 8.0) at room 

 temperature. Genomic DNA was isolated by first 

 powdering tissue under liquid nitrogen with a 

 prechilled mortar and pestle. Approximately 100 mg 

 of powdered tissue were suspended in 500 |aL STE 

 buffer (0.1 M NaCl, 50 mM tris, 1 mM EDTA; pH 

 7.5) and lysed with 25 |aL 20% SDS. Genomic DNA 

 was extracted twice with phenol:chloroform:isoamyl 

 alcohol (25:24:1) and twice with chloroform:isoamyl 

 alcohol (24:1). DNA in aqueous phase was precipi- 

 tated by adding 2.5 volumes of ice-cold absolute etha- 



nol and 0.1 volumes of 3M NaOAC, stored at -20°C 

 for at least two hours, centrifuged for 15 min at 4°C 

 at maximum speed in a microcentrifuge, and rinsed 

 with 70% ethanol. 



PCR amplification for cycle sequencing or restric- 

 tion-enzyme digestion was accomplished by using a 

 suite of PCR primers. The "diagnostic" fragment used 

 in digestions consisted of a single segment, 394-396 

 bp in length, that was amplified by using light-strand 

 primer Cb3RL (CATATTAAACCCGAATGATAYTT) 

 located within the 3' domain of the mitochondrially 

 encoded cytochrome b (cyt b) gene and heavy-strand 

 primer Cb6H (CTCCAGTCTTCGRCTTACAAG) lo- 

 cated within the mitochondrially encoded threonine 

 tRNA (tRNA™!^) gene (Martin and Palumbi, 1993). 

 This fragment was sequenced through the primer 

 sites by using additional primer sets within and out- 

 side the diagnostic fragment. PCR fragments were 

 prepared for sequencing by using the Bio-Rad Prep- 

 a-Gene DNA purification system that removes ex- 

 traneous salts, primers, and small fragments of DNA 

 prior to cycle sequencing. Dideoxy DNA sequencing 

 was performed with the Promega fmol DNA sequenc- 

 ing system by using ■^■^P end-labeled primers. Cycle 

 sequencing reactions consisted of a single two-minute 

 denaturing process at 95°C, followed by thirty cycles 

 of 1 min at 95=C, 30 sec at 64°C, and 30 sec at 72°C. 

 Sequencing reactions were scored on 6% denaturing 



