Hoolihan et al.: Surface mucous as a source of genomic DNA from Istiophoridae and Xiphudae 
341 
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Figure 1 
Representative random amplified polymorphic DNA (RAPD) banding patterns from 
four species: A) Makaira nigricans (blue marlin), B) Tetrapturus albidus (white 
marlin), C) Istiophorus platypterus (sailfish), and D) Xiphias gladius (swordfish) 
for three 10-mer primers (Table 1) tested on each fish for the template DNA from 
surface mucus (SM), and autologous muscle tissue (MT). Lane contents are as fol- 
lows: 1, 100-bp ladder; 2, 5, and 8, negative controls (no DNA); 3 and 4, primer no. 
1; 6 and 7, primer no. 2; and, 9 and 10, primer no. 3. 
genetic analyzer (Foster City, 
CA). Forward and reverse se- 
quences were assembled and 
edited with GeneDoc 2.6.002 
(http://www.psc.edu/biomed/ 
genedoc, accessed 1 June 
2008). To control for cross- 
contamination with other 
billfish or possible parasites 
or microbes, we compared the 
mitochondrial and nuclear se- 
quences derived from surface 
mucous to sequences obtained 
from autologous muscle tis- 
sue. Because autologous mus- 
cle tissue was not available 
for the T. albidus sequencing 
analyses, the mucous sample 
sequences were compared to 
homologous locus sequences 
obtained from reference T. 
albidus tissues available in 
our laboratory. 
Results and conclusions 
RAPD analysis 
Comparable RAPD amplicons 
were derived from surface 
mucous and autologous muscle tissue of M. nigricans, 
T. albidus, I. platypterus, and X. gladius for each of 
the three 10-mer primers tested. Representative RAPD 
banding patterns are illustrated in Figure 1. The nega- 
tive control reactions (minus template DNA) observed 
with each primer confirmed that the amplified genomic 
DNA from both sources was not an artifact of the primer 
concentration. Results indicated that sufficient quanti- 
ties of genomic DNA are available in surface mucous 
from all the species tested. 
Sequencing analysis 
For the three species screened, M. nigricans, T. albidus, 
and I. platypterus, the quality of nuclear and mitochon- 
drial PCR amplifications were comparable. For both the 
nuclear WM13 and the mt ND4 locus, sequences derived 
from a single individual from both sources of genomic 
DNA (mucous and autologous muscle tissue) were found 
to be identical when compared with respect to nucleotide 
base composition (5'-3' and 3'-5' directions), thereby 
demonstrating that the genomic DNA derived from sur- 
face mucous was not due to cross-contamination from 
other billfish or microbes, and in fact originated from 
the fish sampled. Nuclear and mitochondrial sequences 
derived from both sources of genomic DNA are avail- 
able from GenBank under the following accession num- 
bers: T. albidus : ND4 (997bp): FJ809995-FJ809997; 
WM13 (279bp): FJ809988; M. nigricans : ND4 (966bp): 
FJ809991, FJ809992, and FJ809994; WM13 (279bp): 
FJ809986, FJ809987, and FJ809989; I. platypterus-. 
ND4 (1009bp): FJ809990 and FJ809993; WM13 (277bp): 
FJ809984 and FJ809985. 
This study has shown that surface mucous contains 
sufficient quantities of genomic DNA to carry out RAPD 
analyses of istiophorid billfishes and swordfish, as well 
as sequencing applications of istiophorid billfishes. 
These extractions compare favorably to genomic DNA 
extractions from surface mucous reported for freshwater 
species S. formosus (Chansue, 2006), E. lucius, and S. 
trutta fario (Livia et al., 2006). 
The slight differences between some of the mucous 
and muscle RAPD amplification profiles (Fig. 1) may 
be a result of contaminant DNA in the mucous (e.g., 
bacteria, microalgae), or other artifact variations known 
to occur in RAPD studies (Ellsworth et al., 1993). We 
found that the pre-PCR incubation of the template DNA 
master mix with HotStart-IT™ binding protein was 
crucial for preventing mispriming and primer dimer- 
ization that produced these artifact bands (Chou et al., 
1992). In addition, the inclusion of betaine (N, N, N-tri- 
methylglycine) improved band visibility by eliminating 
the smearing attributed to the formation of secondary 
structure, which is caused by G-C rich regions (Henke 
et al., 1997). Importantly, for surface mucous RAPD 
analysis, the RNAase treatment of nucleic acid was a 
necessary step, whereas the inclusion of proteinase K 
was not. 
