NOTES Wilson and Tringali: Isolation and visualization methods for mtDNA studies of clupeids 



613 



sterile, distilled water. Using a Hamilton syringe, 2.0 

 /iL of the digestion buffer was added to the tube and 

 mixed by vortexing, then at least two units of the 

 restriction enzyme were added and also mixed by 

 vortexing. The reaction mixtures were allowed to in- 

 cubate at 37°C for at least 2-3 hours, but sometimes 

 as long as overnight. The reaction was stopped by the 

 addition of 0.4 f^L of 0.5 M EDTA (pH 7.5). 



mtDNA fragment visualization 

 using Hoechst 33258 



Restriction fragments were separated on horizontal 

 (submerged) agarose slab gels with the Hoechst 33258 

 fluorochrome incorporated as described in DeFlaun and 

 Paul (1986). The fluorochrome is a 1:30 dilution of 

 1.5 X 10^-* M solution of Hoechst 33258 with 1 x SSC 

 (i.e., 1:20 dilution of 20 x SSC, Table 1). The gel is 

 created by boiling a 1.1% solution of agarose in dis- 

 tilled Hod containing 10% 10 x TAE (Table 1) buffer 

 until clear. The suspension is allowed to cool to the 

 touch before adding a 1.1% volume of the Hoechst 

 33258 dilution. After casting, the gel is allowed to set 

 for at least 30 minutes in an opaque box to prevent 

 photo-deactivation of the fluorochrome. 



For running, the gel is immersed in a tank buffer of 

 500 mL distilled HoO, 56 mL of 10 x TAE (Table 1), 

 and 5.6 mL of the Hoechst 33258 dilution. Samples 

 containing 10 ^L of digested mtDNA from the reac- 

 tion mixture, 1 f.<L of 10 x TAE, and 2 juh of tracking 

 dye (Bromophenol blue) are then micropipetted into 

 the wells. Outside lanes contained Hind-Ill digested 

 A-DNA standards. 



Gels are electrophoresed at 50 volts for approximate- 

 ly 3 hours, or until the tracking dye covers three- 

 quarters of the length of the gel. The gel apparatus re- 

 mains isolated from light during nmning. Following the 

 run, gels were placed on a UV transilluminator and 

 photographed with a Polaroid MP-4 Land-camera using 

 Polaroid Type 667 black-and-white film. 



Measurement and analysis 

 of mtDNA fragment patterns 



Photographs of the gels were processed on a computer- 

 automated optical pattern-recognition system devel- 

 oped by Biosonics Inc., Seattle, Washington. Fragment 

 sizes were calculated by the global form of the recip- 

 rocal method as described by Elder and Southern 

 (1987). 



Results 



Ovarian tissue from Sardinella aurita and Opistho- 

 nenia oglinum consistently produced visible mtDNA 

 bands in the CsCl/ethidium-bromide gradient by ultra- 

 centrifugation in fewer than 17 hours (Fig. 1), but the 

 testicular tissue did not. If carefully extracted, the 

 mtDNA fraction was sufficiently void of contamination 

 by RNA and nuclear DNA. With the Beckman TL-100 

 it is undoubtedly possible to reduce the time required 

 for ultracentrifugation even further, since higher 

 speeds of up to 100,000 rpm are available using polycar- 

 bonate tubes. 



The only apparent drawback of the fixed-angle rotor 

 over the swinging-bucket rotor is that in the fixed-angle 

 rotor the cross-sectional area of the fluid in the tube 

 is greatest while under centrifugation, but becomes less 

 when the rotor is at rest and least when the tube is 

 upright. Thus, the scab-like material that forms on top 

 of the gradient during centrifugation protrudes down 

 into the gradient when the tube is upright (Fig. 1). 

 However, if the refractive index is adjusted carefully, 

 both DNA bands will form below the protrusion of this 

 scab-like material. Since this material does not crum- 

 ble after centrifugation at 4°C, it can be removed or 

 pushed aside to withdraw the mtDNA. 



The mean yield of mtDNA from fresh 5. aurita 

 gonads stored in MSB-Ca++ for 2 days at 4°C was 

 1.90 + 0.60 ^g mtDNA per g of tissue. The mean yield 

 from fresh S. aurita gonads stored in MSB-Ca+ * for 

 7daysat4°C was 1.18±0.45/.igmtDNApergof tissue. 

 The semifresh 0. oglinum processed after 5 days of 

 storage yielded 1 .01 ± 0.66 /.ig mtDNA per g of ovarian 

 tissue. Isolation of mtDNA from material quick-frozen 

 on liquid nitrogen was also achieved, but yields were 

 reduced by as much as 56%i from that of the semifresh 

 samples. 



Various concentrations of mtDNA were electro- 

 phoresed on Hoechst 33258/agarose gels. The minimum 

 concentration required to produce clear, resolvable 

 restriction fragments (at the 500-base pair (bp) level) 

 was 11.8 ng/^L in 20-^L wells. Fragment mobilities 

 from separate digestions with the two restriction en- 

 donucleases indicated the mtDNA genome size for S. 

 aurita tohe 16,263-16,353 bp, and that of 0. oglinum 

 to be 15,186-15,683 bp. 



Discussion 



The techniques presented here have successfully ad- 

 dressed some of the potential problems associated with 

 performing a large population study using mtDNA. 

 Purification of sufficient quantities of mtDNA to per- 

 form a detailed restriction-site analysis has been 



