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Fishery Bulletin 107(3) 
from three individuals from each of the following species: 
Makaira nigricans (blue marlin), Tetrapturus albidus 
(white marlin), Istiophorus platypterus (sailfish), and 
Xiphias gladius (swordfish). We compared purified DNA 
extracted from mucous and muscle using RAPD, mito- 
chondrial DNA (mtDNA) sequencing, and nuclear DNA 
sequencing analyses. 
Sample preparation and DNA purification 
Surface mucous samples were collected with a poly- 
urethane sponge as described by Schultz et al. (2006) 
and immediately processed, or stored at -80°C for later 
extraction. Each sponge was cut into small pieces, mixed 
with 1500 pL of phosphate buffered saline, and com- 
pressed repeatedly to remove mucous. All fluids were 
centrifuged through a single QIAamp® (Qiagen Inc., 
Valencia, CA) spin column. DNA purification was car- 
ried out by using Qiagen® buccal swab spin protocol 
with the following modifications: 1) a final concentration 
of 1 millimolar (mM) ethylenediaminetetraacetic acid 
(EDTA) was added to the phosphate-buffered saline 
extraction buffer; 2) the proteinase K treatment step 
was eliminated; and 3) an RNase mixture of A and 
T1 enzymes was used to degrade RNA after the final 
DNA purification step (Ambion Inc., Foster City, CA). 
The RNase cocktail enzyme mix was necessary because 
the Qiagen® spin columns copurify RNA and DNA in 
parallel when both are present in a sample. Cold ethanol 
precipitation and inclusion of EDTA in buffers were used 
to reduce nuclease degradation (Dessauer et al., 1996; 
Wasko et al., 2003). 
For muscle tissue, 25 mg were macerated in 180 
pL Buffer ATL (Qiagen Inc., Valencia, CA), and in- 
cubated overnight in a 56°C water bath in the pres- 
ence of 20 pL proteinase K. Purification followed the 
QIAamp® manufacturer’s protocol for tissue. Aque- 
ous samples of nucleic acid (1.5 p L) from mucous and 
muscle extractions were measured for purity with a 
NanoDrop 1000 Spectrophotometer (Thermo Fisher 
Scientific, Wilmington, DE) and showed a range of 1.7 
to 2.0 for the DNA and RNA absorbance ratios (260 
nm:280 nm). 
RAPD analysis 
Three different 10-mer oligonucleotide primers (Table 1) 
of arbitrary sequence (IDT Integrated DNA Technolo- 
gies, Coralville, IA) were tested against surface mucous 
DNA and autologous muscle DNA from three individuals 
of M. nigricans, T. albidus, I. platypterus, and X. gla- 
dius. PCR reactions for RAPD analysis were performed 
in a total volume of 25 pL containing 3 p L extracted 
genomic DNA, 2.5 pL of 10 mM 10-mer primer, 2.5 pL 
of 2.5 mM deoxynucleotide triphosphate (dNTPs), 0.75 
pL of 25 mM MgCl 2 , 0.20 pL Taq DNA polymerase, 2.5 
pL 10x buffer, 5 pL 5M betaine (N, N, N-trimethylgly- 
cine), 2 pL HotStart-IT® binding protein (USB Corp., 
Cleveland, OH), and 6.55 pL distilled water. Pre-PCR 
incubation with the HotStart-IT™ binding protein was 
Table 1 
Nucleotide composition of three 10-mer primers used for 
random amplified polymorphic DNA (RAPD) compari- 
son of DNA extracted from billfish surface mucous and 
muscle tissue, and percent content of guanine and cyto- 
sine (% GC). 
Primer no. 
Nucleotide composition 
% GC 
1 
5'-GTTGCGGGCT-3' 
70 
2 
5'-CAGCCCGGGT-3' 
80 
3 
5'-AGGCCACCGC-3' 
80 
executed at 25°C for four hours to prevent mispriming 
and primer dimerization during amplification (Chou 
et al., 1992). RAPD PCR was performed in an Eppen- 
dorf Mastercycler (Westbury, NY) starting with initial 
heating for 5 minutes at 94°C, followed by 34 cycles at 
94°C for 5 minutes, 42°C for 30 seconds, 72°C for 90 
seconds, and a final extension of 72°C for 10 minutes. 
A negative control (no genomic DNA) was included in 
each PCR set to verify no reagent contamination. The 
PCR products were verified by electrophoresing 5 pL 
in 1.2% agarose gel (ISC BioExpress, Kayville, UT) 
and TAE buffer (pH 8.5) for 60 min at 100 V (60 mA), 
stained with ethidium bromide, and visualized through 
a UV transilluminator. 
Sequencing analysis 
Mitochondrial and single-copy nuclear loci were 
sequenced from three individuals from each of the fol- 
lowing species: M. nigricans, T. albidus, and I. platyp- 
terus. Insufficient samples of mucous and muscle were 
available for sequencing X. gladius. Mitochondrial 
NADH dehydrogenase subunit4 (ND4) was ampli- 
fied by PCR with the primer pair 61F and 1837R and 
cycling parameters outlined in Shivji et al. (2006). All 
amplifications of the anonymous single-copy nuclear 
locus WM13 were performed with the primer pair 
WM13-F and WM13-R developed by Buonaccorsi et 
al. (1999). Nuclear PCR reactions were performed in 
a total reaction volume of 50 p L containing 1 pL of 
extracted genomic DNA, 10 pmol/pL of each primer, 
40 pM dNTPs, lOx PCR buffer, and 1 unit of HotStar 
Taq™ DNA Polymerase (Qiagen Inc., Valencia, CA). 
The PCR thermal profile consisted of an initial heating 
at 95°C for 15 minutes to activate the DNA polymerase, 
followed by 35-40 cycles at 94°C for 1 minute, 1 minute 
at 50°C, and 1 minute at 72°C, with a 5-minute final 
extension step at 72°C. Both nuclear and mitochondrial 
amplifications were performed in a MJ Research PTC- 
200 thermal cycler (Waltham, MA). A negative control 
(no genomic DNA) was included in each PCR set to 
verify that there was no reagent contamination. 
All amplified products were purified by using the 
QIAquick® PCR purification kit (Qiagen Inc., Valencia, 
CA) and sequenced with an Applied Biosystems 3130 
