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Journal of Agrobiology, 25: 89-91, 2008
Alternative storing of DNA and biological samples
using chitosan
Šrubařová P., Civáňová K. et.al.
1
MZLU v Brně
Abstract
The main aim of this study was to find the appropriate way of DNA or biological samples´
storage on chitosan medium (prepared by Food Research Institute in Prague). While
storing these samples on chitosan, it should be possible to keep this material at the room
temperature for a long time. We tried to store the isolated genomic DNA, blood, milk,
meat, sperm, saliva and pieces of ears (all from livestock) on the chitosan in different
thermal conditions to verify the influence of the environment on the sample quality.
Isolation was done using JETQUICK Tissue DNA Spin Kit (GENOMED GmbH) and DNA
was extracted using TE solution. Manipulation with the samples was very difficult.
Comparing results of isolation or extraction DNA in biweekly intervals, we obtained DNA
suitable for further analyses only from archived tissues. To disprove the presumption that
the storing of biological materials (especially DNA) on chitosan is useless, we decided to
continue in this experiment with another form of chitosan and direct the forthcoming
studies to find the fitting way of blood and DNA storing.
Key words: Chitosan, DNA storage, DNA isolation
Introduction
Storing of DNA and biological samples in a
stabilized state at room temperature and their
further applying directly to the studies and
analyses is an important object in the field of
molecular genetics and medicine. Ideal method
for storing of DNA and biological samples
should be simple, cost effective, without DNA
degradation and without any problems to use
DNA and biological samples for next analysis,
and last but not least, a range of samples
should be stored in a limited space.
A lot of inventions relate to DNA storing
methods with the aim to preserve it in a
stabilized state at room temperature for an
extended time. In this type of storing, the
unwavering quality of DNA is very important
factor for the future analyses. There are many
commercial products available but they have
limitations to its effectiveness. Mostly, the
producers advise to store frozen DNA on the
special medium that makes the storage
financially more exacting.
Chitosan – a biologically produced polymer
seems to be very suitable for storing of DNA
and biological samples. It is widely used
especially in the field of medicine because of its
stability, biodegradability and suitability on a
living body (Woo-Chul et al., 2007). This
polysaccharide comprising copolymers of
glucosamine and N-acetyl-glucosamine has
great potential in food industry and
biotechnology applications because of its
unique cationic character. It has been shown to
be an effective coagulating agent in wastewater
treatment and recovery of lipids and proteins
from plant processing food wastes including
dairy wastewater, as well as in precipitation of
casein micelles (Casal et al., 2006). Because of
these properties it can be used safely by the
dairy processing industry, too.
Actually, chitosan is an effectual derivate
from chitin, polysaccharide with positive charge
what cause its very strong binding properties. It
was prepared for the first time in 1859 by prof.
Rouget. First patent was published in 1920.
Since this time, hundreds of patents were
published. Czech scientists have many
significant inventions in the field of using
chitosan, especially Food Research Institute in
Prague (bok, 2006). In most cases, chitosan is
extracted from crushed shells of crabs or
oysters but Czech scientists can get this
material from waste. Production of chitosan
using this procedure is significantly cheaper
and raw material is still available. Chitosan can
be used also against obesity, in cloth coloring
or like an implant which can be transfer into
patient’s body to cure damage (Mařík, 2006).
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Journal of Agrobiology, 25: 89-91, 2008
Material and methods
Chitosan was prepared by scientists in
Food Research Institute in Prague. Biological
samples and DNA samples were stored on
chitosan. A total of 30mg of meat, part of ears;
20µl of blood, milk, saliva; 10µl of sperm and
5µl of isolated DNA was transferred to three
special plates (microtitrate plate with affixed
chitosan), each to different hole in parallel way.
Meat and pieces of ears were grind before
storing. To compare the influence of the storage
environment, tree plates with the same samples
were prepared in the same way and stored in
different conditions – at room temperature, in a
freezer and in a thermostat at 72°C.
DNA from biological samples stored in
chitosan was isolated in several time periods by
JETQUICK Tissue DNA Spin Kit (GENOMED
GmbH) and DNA was extracted using TE
solution (10 mM Tris-HCl, pH = 7,5; 0,1 mM
EDTA). After electrophoresis on 1.5% agarose
gel (10 min, 100V), DNA isolated from all
samples was visualized using ethidiumbromide
staining.
For validating quality of isolated DNA, PCR
(polymerase chain reaction) was done. The
880bp amplicon of MC3R gene (gene for
melanocortin 3 receptor) was chosen. The
amplification for blood, pork meat and ear
samples were carried out in a final volume of
25 µl in a reaction mixture containing 8,5 µl of
water, 12,5 µl of PPP Master Mix (150 mM TrisHCl, pH 8,8, 40 mM (NH4)2SO4, 0,02% Tween
20, 5 mM MgCl2, 400 µM dATP, 400 µM dCTP,
400 µM dGTP, 400 µM dTTP, 100 U/ml Taq
Purple DNA polymerase, monoclonal anti-Taq
DNA polymerase (38 nM)), 1 µl of MC1A
primer, 1 µl of MC1B primer and 2 µl of DNA.
The amplification was run in a PTC-100TM
termocycler (MJ Research, Inc., USA) using the
following conditions: initial activation step at 95°
for 2 min, followed by 30 cycles at 95° for 20 s,
at 65° for 20 s and at 68° for 7 min. PCR
products were visualized after electrophoresis
(20 min, 100 V) on 1,5% agarose gel with
ethidiumbromide.
Results and discussion
The purpose of the present study was to
develop new method for simple and cost
effective storing of biological samples and DNA
without DNA degradation. Binding medium chitosan was prepared in Food Research
Institute in Prague and DNA was isolated in
several time periods using method already
proved to be useful at our workplace.
Manipulation with biological samples stored
in
chitosan
differs.
More
complicated
manipulation was with samples from the freezer
and the thermostat because the chitosan lost
compatibility and it was very difficult to take the
whole sample out of the plate. The easiest work
was with samples stored at the room
temperature.
DNA isolation and DNA extraction was
done in 2 weeks intervals. The quality of
samples was checked by gel electrophoresis on
agarose gel after each DNA isolation or DNA
extraction. The results differed a lot. The DNA
isolation from milk, saliva and sperm failed and
no DNA was extracted from chitosan. After 2nd
isolation (4 weeks later), the isolation from
blood samples was unsuccessful, too except
the samples stored in the thermostat.
The amount of DNA isolated form meat and
pieces of ears was quite high.
The gene for melanocortin 3 receptor
(MC3R) was chosen because PCR products
are relatively long (880 bp) and the degradation
of
DNA
can
be
verified
by
PCR
accomplishment. The amplification of DNA
fragments was successful only in the samples
with DNA from meat and piece of ears (Figure
1). For length validation of DNA fragments, two
size markers– M23 and M100 (MBI Fermentas,
Lithuania) were used.
This confirmed that the best quality DNA
was obtained by isolation from tissues (meat,
pieces of ears) and that the storage of other
samples (blood, isolated DNA, saliva and
sperm) on chitosan and DNA isolation from
them seem to be not very convenient.
When
comparing
different
storing
conditions, the room temperature is good
because of manipulation with samples.
Comparing the outcomes between room
temperature (approx. 25 ºC), -20 ºC and 72 °C
according to DNA amount, the best results were
obtained from samples stored in thermostat.
Figure 1: PCR products
Note: Sample 1 is PCR product of DNA isolated from blood
stored at the room temperature after 2 weeks storing;
Sample 2 is PCR product of DNA isolated from meat stored
at the room temperature after 2 weeks storing; Sample 3 is
PCR product of DNA isolated from blood stored in the
temperature-controlled oven after 4 weeks storing; Sample
4 is PCR product of DNA isolated from piece of ear stored
at the room temperature after 4 weeks storing; Sample 5 is
PCR product of DNA isolated from blood stored in the
temperature-controlled oven after 6 weeks storing; Sample
6 is PCR product of DNA isolated from piece of ear stored
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Journal of Agrobiology, 25: 89-91, 2008
in the freezer after 6 weeks storing; Sample 7 is a reference
sample.
The main problem with this type of
biological samples or DNA storing is in the
initial amount of the sample. Chitosan cannot
adsorb so much fluid so the sample volume that
can be stored in chitosan is limited.
Conclusion
Although the literature sources indicate that
chitosan seems to be a good medium for
storing of DNA, it can be concluded that the
method described in this article is unsuitable for
DNA archive. However, even if it is very difficult
to prepare appropriate form of chitosan for good
manipulation we decided to do whole
experiment with another form of chitosan in our
forthcoming studies, all in cooperation with
Food Research Institute in Prague.
This work was supported by the Ministry of
Agriculture of the Czech Republic (project no.
1G58073).
Literature
Casal, E. et al. Use of Chitosan for Selective
Removal of β-Lactoglobulin from Whey. J. Dairy
Sci. 2006, no. 89, s. 1384-1389.
Mařík, M.
Nové
lepidlo
pro
chirurgy.
Hospodářské noviny. 14.6.2006. Dostupný
z WWW:<http://hn.ihned.cz/2-18673240500000_d-2b>.
Woo-Chul, M., et al. (Goodgene Inc.): Method
for storing DNA using chitosan, and products
using the methods. US 0254294A1 (2007);
http://www.freepatentsonline.com/y2007/02542
94.html (9.4.2008).
Corresponding author:
Ing. Šrubařová Petra
Department of animal morphology, physiology
and genetics
Mendel University of Agriculture and Forestry in
Brno
Zemědělská 1, 613 00 Brno
Czech Republic
Email: [email protected]
Telephone: +420 545 133 377
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