Improved Methods for Isolation of 

 Fish mtDIMA by Ultracentrifugation and 

 Visualization of Restriction Fragments 

 Using Fluorochrome Dye: Results 

 From Gulf of Mexico Clupeids 



Raymond R. Wilson Jr. 

 Michael D. Tringali 



Department of Marine Science. University of South Florida 

 St Petersburg, Florida 33701 



The isolation of mitochondrial DNA 

 (mtDNA) for studies of restriction- 

 site polymorphisms among fish pop- 

 ulations is achieved by either of two 

 general methods. The methods are 

 those of Lansman et al. (1981) 

 which uses ultracentrifugation in 

 cesium-chloride/ethidium-bromide 

 gradients to separate mtDNA from 

 nuclear DNA, and Chapman and 

 Powers (1984) which uses low-speed 

 centrifugations and organic extrac- 

 tions to precipitate mtDNA. Al- 

 though the method of Chapman and 

 Powers (1984) results in mtDNA of 

 lesser purity, it apparently requires 

 fewer hours to isolate mtDNA from 

 a greater number of specimens than 

 the method of Lansman et al. 

 (1981), and avoids the high cost of 

 purchasing and servicing a typical 

 ultracentrifuge. 



The technique chosen for visual- 

 ization of restriction fragments de- 

 pends upon the amount of mtDNA 

 obtainable from the specimens of in- 

 terest and its purity. Neither of the 

 commonly reported techniques, such 

 as end-labeling and ethidium-bro- 

 mide staining, is specific for mtDNA. 

 Since end-labeling is about 1000 x 

 more sensitive, a more highly puri- 

 fied preparation is required than for 

 ethidium bromide. Radio-labeled hy- 

 bridization probes are highly spe- 

 cific for mtDNA as well as very sen- 

 sitive, but require many steps for 

 preparation (Maniatis et al. 1982) 



and must be periodically remade. 

 Biotinylation may provide longer- 

 lived hybridization probes (Graves 

 et al. 1990) but making them also 

 requires several steps. 



Here we introduce a new com- 

 bination of isolation and visualiza- 

 tion methods for mtDNA studies 

 which incorporate attributes of some 

 of the above techniques and which 

 may aid investigators developing 

 plans to begin mtDNA studies. Our 

 approach was developed for poten- 

 tial use in a population study of 

 Spanish sardine Sardinella aurita, 

 as well as of other clupeids of the 

 western Atlantic, while mindful of 

 the need to process statistical sam- 

 ple sets. Incorporated in this pre- 

 sentation of methodology is a brief 

 account of our initial findings on 

 Gulf clupeids. 



Materials and methods 



Sample collection and 

 preparation 



Sardinella aurita and Opisthonema 

 oglinum were collected aboard com- 

 mercial purse seine vessels in the 

 nearshore regions of central-west 

 and north Florida between April 

 and August 1989. Male and female 

 gonads from these samples were 

 excised fresh or semifresh and im- 

 mediately stored in 10 mL of cold 



MSB-Ca++ (Table 1) for up to 7 

 days at 4°C. The tissue was blotted 

 dry, weighed, minced, and then 

 homogenized in 2 to 3 volumes of 

 cold MSB-Ca+ + by 5 strokes of a 

 chilled 15-mL Dounce homogenizer. 

 The homogenates were transferred 

 to chilled 50-mL polyethylene cen- 

 trifuge tubes after addition of di- 

 sodium EDTA (0.2 M, pH 7.5) to a 

 final concentration of 10 mM. 



The homogenate was centrifuged 

 at 800 X g for 10 minutes at 4°C 

 after which the supernatant was de- 

 canted and respun. These two spins 

 remove nuclei and cellular debris. 

 Mitochondria were then pelleted by 

 centrifugation at 20,000 x g for 

 20 minutes at 4°C. The pellet was 

 washed with 10-20 mL of cold 

 MSB-EDTA (Table 1) and repel- 

 leted by centrifugation for 15 min- 

 utes at 20,000 X g. 



The pellets were suspended in 2 

 mL of cold STE (Table 1) and the 

 mitochondrial membranes dissolved 

 by adding 0.10 mL of 25% SDS, 

 vortexing briefly and incubating at 

 37°C for 5-8 minutes. The entire 

 lysate was then transferred to 3.0- 

 mL Beckman polyallomer or poly- 

 carbonate centrifuge tubes contain- 

 ing enough cesium chloride (Sigma 

 #C-3032) to produce a concentration 

 of 1 . 1 g CsCl per mL of lysate as in 

 Lansman et al. (1981). To this was 

 added 0.133 mL of 0.025 M ethidi- 

 um bromide. The solution was then 

 mixed by inverting several times, 

 and the refractive index of each 

 tube adjusted to 1.389 at 20°C with 

 CsCl or STE. Solution density at 

 this Rf causes the DNA to float 

 lower in the tube than at the Rf 

 given by Lansman et al. (1981). 



Reference to trade names does not imply en- 

 dorsement by the National Marine Fisheries 

 Service, NOAA. 



Manuscript accepted 27 April 1990. 

 Fishery Bulletin, U.S. 88:611-61.5. 



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