Presentation on theme: "A quick and cheap new DNA extraction method from lymnaeids snails Reference: - Walsh PS, Metzger DA, HIguchi R (1991) Chelex 100 as a medium for simple."— Presentation transcript:
A quick and cheap new DNA extraction method from lymnaeids snails Reference: - Walsh PS, Metzger DA, HIguchi R (1991) Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechnology 10:506-513. Caron Y., Lekimme M., Losson B. Faculty of Veterinary Medicine, Department of Infectious and Parasitic Diseases, University of Liège, 20 Boulevard de Colonster, B43a, 4000 Liège, Belgium. Email: firstname.lastname@example.org@ulg.ac.be Tel: +32 (0) 4 366 40 09 Xth European Multicolloquium of Parasitology from Satellites to Microsatellites Paris – August 24th – 28th, 2008 Introduction: During large epidemiological surveys efficient tools are needed to assess the vectorial capacity or the accurate identification of lymnaeid snails responsible for the development and release of the larval stages of Fasciola sp. DNA extraction need to be fast, safe, cheap and to provide good quality of DNA for further analyses (PCR, cloning, sequencing). The chelex 100 is a chelating ion exchange resin first described in a DNA extraction protocol (Walsh et al, 1991). It is used with different tissues (blood, semen) and organisms (human, insects). This short communication aims to compare the classical phenol/chloroform with chelex-based DNA extraction methods for lymnaeid snails. Materials, methods and results: The DNA of one hundred Galba truncatula collected in july 2007 in the region of Boutheldja (Algeria) was extracted following two DNA extraction methods: -Classical phenol/chloroform-based DNA extraction method: The material was mechanically disrupted in 100 µl of water and incubated at 50°C overnight in 100 µl of lysis buffer (50 mM TrisHCl pH8, 100 mM NaCl, 50 mM EDTA, 0.8% SDS) and 200 µg/ml proteinase K. Thereafter, 100 µl of phenol/chloroform/isoamyl-alcohol (25/24/1) was added to the initial mix. The mixture was placed on a vortex for 1 min and centrifuged at 12 000 g for 10 min. The supernatant was transferred to a microcentrifuge tube containing 100 µl of chloroform/isoamyl-alcohol (24/1), gently mixed and centrifuged at 12 000 g for 10 min. The supernatant was transferred into another microcentrifuge tube containing 250 µl of ethanol 100% and 50 µl of sodium acetate (3M), incubated overnight at room temperature and then centrifuged at 12 000 g for 30 min. The pellet was washed with 250 µl of ethanol 75% and centrifuged at 12 000 g for 10 min, dried for several minutes, rehydrated and stored at -20°C. -Chelex-based DNA extraction method: The material was mechanically disrupted in 100 µl of water and incubated one hour at 56°C and 30 min at 95 °C in 100 µl of chelex 100 5% (Bio-Rad). Then the mixture was centrifuged at 14 000 g for 3 min. The supernatant was collected and stored at -20°C.The concentration and the 260/280 wavelength ratio of both extracted DNA series from the two techniques were mesured with a spectrophotometer (Nanodrop). Moreover the time required for each techniques and the cost was also evaluated. The results are shown in the table below. DNA concentration (ng/µl) Wavelength ratio (260/280) Time processing (hours) Cost (€/snail) Phenol/chloroform56.3 ± 33.11.96 ± 0.21*240.5 Chelex80.0 ± 25.21.63 ± 0.07*50.2 * Statistically significant (p<0.05) Discussion and conclusions: This is the first time that the chelex 100 is used to extract DNA from snails. The DNA concentrations are comparable with the two techniques, contrary to the wavelength ratio. Furthermore the quality of chelex extracted DNA was tested in PCR and cloning/sequencing procedures with excellent results (in preparation). The chelex-based DNA extraction is very simple, fast (5 versus 24 hours), and does not require organic solvents and multiple tube transfers. The extraction of snail DNA using Chelex 100 is, particularly regarding time processing and low cost, at least as efficient as the proteinase K and phenol/chloroform extraction making this technique an excellent procedure to analyse large samples. This technique will be used to assess the epidemiology of Fasciola hepatica in Belgium in several lymnaeid snails species. Acknowledgment: We are very gratefull to Dr. Souad Righi and Pr. Ahmed Benakhla of the University Center of El-Tarf (Algeria) for the providing of the snail sample.