292 



Yellow (.Perca f/avescens) and Eurasian 

 iP. fluviatilis) perch distinguished in 

 fried fish samples by DNA analysis 



Rex M. Strange 



Carol A. Stepien (contact author) 



Email address for C, A. Stepien: CarolStepieniaiutoledoedu 



Great Lakes Genetics Laboratory 



Lake Erie Center and Department of Environmental Sciences 



The University of Toledo 



6200 Baysfiore Road 



Toledo, Ohio 43618 



DNA techniques are increasingly 

 used as diagnostic tools in many 

 fields and venues. In particular, a 

 relatively new application is its use 

 as a check for proper advertisement 

 in markets and on restaurant menus. 

 The identification of fish from mar- 

 kets and restaurants is a growing 

 problem because economic practices 

 often render it cost-effective to sub- 

 stitute one species for another. DNA 

 sequences that are diagnostic for 

 many commercially important fishes 

 are now documented on public data- 

 bases, such as the National Center for 

 Biotechnology Information's (NCBI) 

 GenBank.i It is now possible for most 

 genetics laboratories to identify the 

 species from which a tissue sample 

 was taken without sequencing all the 

 possible taxa it might represent. 



We were contacted by reporters 

 from a news agency, who were in- 

 terested in determining whether yel- 

 low perch "fish fries" in their local 

 restaurants were, in fact, local yel- 

 low perch (Perca flavescens Mitchell) 

 taken from the Great Lakes. In re- 

 cent years it has become economical- 

 ly desirable to substitute Eurasian 

 perch (P. fluviatilis Linnaeus) or 

 some other species for yellow perch 

 because of the decline of stocks of 

 yellow perch in the Great Lakes 

 and because of rising prices, both 

 of which raise truth-in-advertising 

 questions. Such cases of substitution 

 usually involve replacement with 

 less expensive fish species, which 

 are difficult to detect in fillets (see 

 Ward, 2000). 



We agreed to attempt to identify 

 the fish using DNA techniques be- 

 cause the Great Lakes Genetics Lab- 

 oratory has several on-going projects 

 involving evolutionary genetics of 

 yellow perch and other percids (e.g., 

 Faber and Stepien, 1997; Ford and 

 Stepien, 2004). However, instead of 

 the fresh frozen fillets that we ex- 

 pected, the reporters sent us breaded 

 and deep fried fish fillets taken from 

 served dinners. We decided to at- 

 tempt to identify the species of the 

 fish, despite our initial misgivings as 

 to whether useful amounts of DNA 

 could be extracted from deep-fried 

 material. In the present study, we 

 outline a procedure with molecular 

 tools and an analysis that allows 

 the identification of any species for 

 which sequences are documented on 

 GenBank.i 



Materials and methods 



We removed approximately 10 mg of 

 muscle tissue from below the bread- 

 ing of each fried fillet, submerged the 

 tissue in an ethanol (95%) wash, and 

 allowed the wash to sit overnight at 

 room temperature. No other attempt 

 was used to remove any residual 

 cooking oil. Samples were then air- 



GenBank. 2006. National Center 

 for Biotechnology Information (NCBI), 

 National Institutes of Health, 8600 

 Rockville Pike, Bethesda, MD 20894. 

 Website: http://www.ncbi.nim.nih.gov 

 (accessed 14 February 2006). 



dried before digestion with protein- 

 ase K. After complete digestion, DNA 

 was isolated by using standard phe- 

 nol-chloroform extractions, alcohol 

 precipitation, and two washes with 

 70% ethanol. From experience, we 

 found that the final pellet of DNA was 

 smaller than the DNA yield we would 

 expect from uncooked material; there- 

 fore we suspended the pellet with 30 

 ViL (microliters) of dd (deionized dis- 

 tilled) H^O rather than the 100 /(L we 

 typically use. 



The polymerase chain reaction 

 (PCR) was used to amplify a frag- 

 ment (approximately 400 base pairs) 

 of the mitochondrial cytochrome b 

 gene with the following universal 

 primers described by Palumbi (1996): 

 L14724 (5'-GTG ACT TGA AAA ACC 

 ACC GTT G-3') and Kocher et al. 

 (1989): H15149 (5'-TGC AGC CCC 

 TCA GAA TGA TAT TTG TCC TCA- 

 3'). We chose this particular frag- 

 ment for two reasons: 1) cytochrome 

 b is commonly used in systematic 

 studies of fishes and a wide variety 

 of fish taxa are documented in the 

 GenBank' data base, including per- 

 cids (e.g.. Song et al., 1998; Near, 

 2002; Sloss et al., 2004); and 2) our 

 previous experience has shown that 

 PCR amplification of smaller frag- 

 ments is often more successful than 

 amplification of larger fragments, es- 

 pecially when dealing with degraded 

 samples such as extracts from deep- 

 fried fillets. 



The PCR mixture consisted of a to- 

 tal volume of 50 //L, with concentra- 

 tions of 1.5 mM (milli Molar) MgClg, 

 1.0 /^M (micro Molar) of each primer, 

 and 1.0 U (units) of Taq polymerase. 

 Amplification parameters consisted of 

 an initial denaturation at 94°C for 2.5 

 min, followed by 35 cycles of denatur- 

 ation (94°C, 1 min), primer annealing 

 (52°C, 1 min), and polymerase exten- 

 sion (72°C, 1 min). A final extension 

 at 72°C for 7 min was included to 

 reduce the number of partial strands. 

 Amplification products were then pu- 

 rified by running the entire product 



Manuscript submitted 16 June 2006 

 to the Scientific Editor's Office. 



Manuscript approved for publication 



6 September 2006 by the Scientific Editor. 



Fish. Bull. 105:292-295 (2007). 



