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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). 89 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 90 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 91