Fisheries Science 62(5), 727-730 (1996)

Tissue Preservation and Total DNA Extraction from Fish Stored

at Ambient Temperature Using Buffers Containing
High Concentration of Urea

Takashi Asahida,*1 Takanori Kobayashi,*2 Kenji Saitoh,*3 and Ichiro Nakayama*4

*1Tohoku National Fisheries Research Instit
ute, Shinhama, Shiogama, Miyagi 985, Japan
*2National Research Institute of Aquaculture
, Nansei, Mie 516-01, Japan
*3Tohoku National Fisheries Research Institute Hachinohe Branch
, Same, Hachinohe,
Aomori 031, Japan
*4National Research Institute of Aquaculture Tamaki Branch
, Tamaki, Mie 519-04, Japan
(Received January 12, 1996)

We have developed a high concentration urea containing buffer (TNES-Urea: 6 or 8 mt urea; 10 mM

Tris-HCl, pH 7.5; 125 mM NaCl; 10 mM EDTA; 1% SDS) for DNA extraction by modifying the cell ly
sis buffer for DNA isolation, and we found this buffer is suitable for long-term preservation of tissue
samples from fish at ambient temperatures and for DNA extraction from fish that are rich in cellular en
donucleases. Tissue samples from the Japanese flounder Paralichthys olivaceus and Atlantic herring

Clupea harengus were preserved for periods ranging from 1 month to 3 years and for the Atlantic her
ring transported from Sweden to Japan in TNES-Urea buffer at ambient temperature (10-36•Ž). The
total DNA for each fish was extracted from the muscle or liver tissue which had been preserved for

periods ranging from 1 month to 3 years. The DNA yield was 0.5-2.6 ƒÊg of total DNA/mg tissue. All
DNA from preserved tissues was suitable for DNA analyses, e.g. Polymerase Chain Reaction (PCR)
technique, Southern blot analysis and Random amplified polymorphic DNA (RAPD) analysis. The
TNES-Urea buffer provides a convenient method of tissue preservation and DNA extraction and offers
an alternative to previous methods which require protocols that are restrictive in some field settings.

Key words: tissue preservation, urea, total DNA extraction, PCR, RAPD analysis, Southern blot
analysis, Paralichthys olivaceus, Clupea harengus

The preservation and transportation of tissue samples

for DNA analysis are normally carried out by freezing on Materials and Methods
dry ice. However, dry ice is often difficult to obtain in re
mote locations, tropical regions and undeveloped coun Fish Samples
tries, and is not suitable for transportation over long We used Japanese flounder, Paralichthys olivaceus to ex
periods because it sublimates rapidly. Furthermore, dry amine tissue preservation and some DNA analyses, and At
ice is restricted on some forms of public transportation. lantic herring Clupea harengus to examine effects of trans
Although nucleic acids are extremely stable molecules, portation from Sweden to Japan and DNA analysis.
they are easily degraded by cellular endonucleases, e.g.
DNase. Moreover, some fish have powerful cellular en Tissue Preservation and DNA Extraction
donucleases which prevent DNA extraction. Inhibition or We developed the following TNES-Urea buffer: 6 or 8 m
denaturation of these enzymes is needed for successful tis urea; 10 mM Tris-HCl, pH 7.5; 125 mM NaCl; 10 mM
sue preservation and DNA extraction. EDTA; 1% sodium dodecyl sulfate (SDS), by modifying
Proebstel et al.1) reported a tissue preservation method the cell lysis buffer for isolation of total cellular DNA
using a dimethyl sulfoxide (DMSO) salt solution. They from animal tissues.2) 50-100 mg of muscles or liver tissue
examined samples that were transported at ambient tem was removed from each fish and preserved in 400-600 ƒÊl of

peratures (10-32•Ž) and stored under normal refrigera the buffer at ambient temperature (10-36•Ž) for periods
tion conditions (5•Ž) for 25-45 weeks, but they did not ranging from 1 month to 3 years. During the shipping proc
test long-term storage of samples at ambient temperatures. ess, we kept the tissue samples in parafilm-sealed
Moreover, their protocol requires the solution to be microtubes. For tissue digestion, 0.8 mg of Proteinase K
changed prior to DNA extraction. Here, we report a buffer was added and the mixture incubated for 8-16 hours at
which we have developed and found suitable for long-term 37•Ž or for more than 3 days at room temperature (20-25•Ž
preservation of tissue samples at ambient temperatures ). The mixture was extracted with phenol-chloroform
and for total DNA extraction from fish rich in cellular en (1:1). After the ether extractions, the DNA was precipitat
donucleases . ed with 2 volumes of ethanol in 0.3 m NaCl and then
resuspended in TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM
EDTA).
728 Asahida et al.

DNA Analysis
We amplified the D-loop containing region of mitochon
drial DNA (mtDNA) by Polymerase Chain Reaction

(PCR). PCR conditions were performed in volumes of 50

ƒÊ
l containing 10 mm Tris-HCl, pH 8.3; 50 mm KCl, 1.5
mm MgC12, 0.001% gelatin, 200ƒÊM each of dATP, dCTP,
dGTP and dTTP (Takara), 0.2 ƒÊm each of primer 1 and 2,
2.5 unit of Taq DNA polymerase (Takara), and 10-500 ng
of template DNA. The PCR primers for amplification of
the 2.5 kb segment were modified from L15996 by Vigilant
et al. (5•Œ-TCACCC (C/T) T (A/G) (A/G) CT (A/C)

CCAAAGC-3•Œ)3) for the L-strand and complementary of

L1091 by Kocher et al.4) for the H-strand. The PCR

primers for amplification of the 1.1 kb segment were the

same as above for the L-strand and 5•Œ-TGAGACTAT
Fig. 1. Total DNA of Japanese flounder obtained from TNES-Urea
TTACTTGCACGC-3•Œ for the H-strand. The H-strand
buffer-preserved tissue.
primer was designed from sequence data of the D-loop The DNA in lanes 2-7 was extracted from muscle tissue preserved
region (Saitoh et al., unpublished). Oligonucleotide for 1 month, 3 month, 6 month, 1 year, 2 years and 3 years, respec

primers were synthesized by cyanoethyl-phosphoramidite tively. Lane 1 contains Hind ‡V digests of ƒÉ phage DNA as a molecu

chemistry on a Beckman Oligo 1000 DNA synthesizer. Am lar weight marker.

plification was performed in a Perkin Elmer GeneAmp

PCR System 9600 DNA Thermal Cycler programmed for
30 cycles of 30 sec at 94•Ž, 30 sec at 55•Ž and 2 min at 72•Ž,
with 5 min at 72•Ž for the final extension step. The
PCR products were digested by 10-15 units of restriction
endonucleases under the manufacturer's recommended
conditions. The restricted PCR products were analyzed by
electrophoresis in 4% Nusieve 3:1 agarose (Takara) gels
and detected by staining with ethidium bromide (EtBr).
We also analyzed restriction patterns of the entire mtDNA
of Japanese flounder using Southern blot hybridizations.
The total DNA extracts underwent enzyme digestion,
0.8% agarose gel electrophoresis in TAE, transfer onto a
nylon membrane (Hibond-N, Amersham) and hybridiza
tion with appropriate probes labeled with digoxigenin

(Boehringer Mannheim). Japanese flounder mtDNA,

purified by two rounds of CsCl ultracentrifugation,sl was Fig. 2. PCR products of mtDNA of Japanese flounder.

used as the probe for detection of the entire mtDNA. 2.5 kb (lanes 2-4) and 1.1 kb (lanes 5-7) segments of the D-loop

Hybridization and immunological detection were containing region of the mtDNA were amplified. Lane 1 contains Sty

I digests of ă phage DNA as a molecular weight marker.

preformed using the supplier's recommendations. Ran
dom amplified polymorphic DNA reactions6) were per
formed with arbitrary primers (primer A, 5•Œ-AG
GTCACTGA-3•Œ; B, 5•Œ-TGGTCACTGA-3•Œ; C, 5•Œ
TCACGATGC-3•Œ).6) Amplification was performed in a
Perkin Elmer Gene Amp PCR system 9600 DNA Thermal
Cycler programmed for 40 cycles of 1 min at 94•Ž, 1 min
at 40•Ž and 2 min at 72•Ž. Amplification products were
analyzed by electrophoresis in 2% agarose gels and detect
ed by staining with EtBr.

Results

The total DNA extracted from Japanese flounder was

obtained from muscle or liver tissue which had been

preserved in the TNES-Urea buffer for periods ranging

from 1 month to 3 years (Fig. 1). Also, the total DNA of
Atlantic herring was obtained from muscle tissue which
Fig. 3. Dpn ‡U (lanes 2-11) and Taq ƒ¿ I (lanes 12-21) digestion patterns
had been transported from Sweden to Japan and stored at
of PCR products of mtDNA from Japanese flounder.
ambient temperature (20-36•Ž). The DNA yield was 0.5
1.1 kb segments of D-loop containing region of mtDNA were am
2.6ƒÊg of total DNA/mg tissue even although it was ex
plified and digested with 12 restriction endonucleases. Lanes I and
tracted from extensively (3 years) preserved tissue. 22 contains 100 base pair ladder DNA molecular weight markers
2.5 kb and 1.1 kb segments of the D-loop containing (Pharmacia Biotech).
 Tissue Preservation Technique for DNA Analysis 729

Fig. 6. Total DNA of Japanese flounder.

The DNA in lanes 2-10 was extracted from muscle tissue

preserved in 0 M, 1 mt, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M and 8 M urea con

Fig. 4. Hind ‡V digestion patterns of entire mtDNA of Japanese taining buffer, respectively. Lane I contains Hind ‡V digests of d
flounder analyzed by Southern blot hybridization. phage DNA as a molecular weight marker.
Lane 1 and 11 contains Sty I digests of ă phage DNA as a molecu

lar weight marker.

perform 20 Southern blot assays.

Figure 5 shows the results of an experiment in which
RAPD analysis was performed on genomic DNA from
preserved tissues of Japanese flounder and Atlantic her
ring. Polymorphic DNA fragments were observed by
RAPD analysis using some primers.

Discussion

Although DNA analysis is an indispensable method for

fish biology, it is difficult to apply it to samples from re
mote locations. We have demonstrated that this tissue
preservation technique can be used successfully in fish biol
ogy. We found that the TNES-Urea buffer is suitable for
transportation and long-term preservation of tissue sam
ples at ambient temperature. This preservation technique
offers advantages over previous methods which require
protocols that may be restrictive in some field settings.
The ability of this buffer to preserve tissue samples de
Fig. 5. RAPD patterns of genomic DNA of Japanese flounders (lanes pends on the concentration of urea used. More than 4
mole (M) urea containing buffer shows good ability for tis
2-4, primer A) and Atlantic herrings (lanes 5-7, primer B).
Lane 1 contains 100 base pair ladder DNA molecular weight mar sue preservation (Fig. 6). Especially high concentrations of
ker (Pharmacia Biotech). urea (6-8 M) containing buffer appear most suitable for
long-term storage at high ambient temperatures (more
than 30•Ž). There was no degradation of the total DNA ex
tracted from tissues when preserved in 6-8 M urea contain
region of the mtDNA of Japanese flounder were amplified ing buffer for up to 3 years at ambient temperatures (10
by PCR (Fig. 2). Some haplotypes were observed in the 36•Ž). Moreover, the total RNA that was extracted from
analysis of PCR products by restriction endonucleases long-term preserved tissue was also detected by agarose gel
(Fig. 3). All DNA extractions from the TNES-Urea buffer electrophoresis with EtBr staining. Castelli et al.7) reported
Preserved tissues yielded template DNA which was active some results of DNA fingerprinting in fish using 8 M urea
in PCR. No contaminating DNA was detected in the nega containing buffer for tissue homogenization. Although
tive controls for extraction or PCR amplifications. urea is a denaturant of DNA, it did not influence the
Figure 4 shows Hind ‡V digestion pattern of entire mtD results of some DNA analyses. However, in the case of us
NA of Japanese flounder as analyzed by Southern blot ing the 8 M urea containing buffer, it was difficult to
hybridization. We have mapped the cleavage sites of recover the aqueous phase when the mixture was extracted
twelve restriction endonucleases in the mtDNA of with phenol-chloroform, because the aqueous phase was
Japanese flounder (data not shown). The total DNA ob very viscous. To avoid this problem, the mixture should be
tained from 50-100 mg of preserved tissue was sufficient to incubated with Proteinase K for more than 48 hours at
730 Asahida et al.

37•Ž or more than 7 days at room temperature. It is easier niques of DNA analysis, e.g. PCR techniques, Southern
to recover the aqueous phase when using a 4 to 6 M urea blottings and RAPD analysis offers advantages when work
containing buffer than using 8 M urea containing buffer. ing with some specimens that live in remote areas or when
We recommend using 8 M urea containing buffer for preser the transport of frozen samples is not possible.
vation at high ambient temperatures, and 6 M urea for ordi
nary preservation of tissue samples and for tissue transpor Acknowledgments We would like to thank Mr. Jun Aoyama and Dr.

tation. TNES-Urea buffer is suitable for DNA extraction Toru Kitamura for technical assistance, Dr. Keisuke Takada and Mr.
Yoshimasa Aonuma for provided information about DNA extraction
from fish that are rich in cellular endonucleases such as
from formalin fixed tissues, and Drs. Yoh Yamashita and Chris Norman
Japanese flounder, because urea is an inhibiter of cellular
for helpful discussion and critical reading of the manuscript.
endonucleases. Also, this buffer is more suitable than an
ordinary buffer for DNA extraction from tissue, because
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