Evaluation of three methods for high throughput extraction of DNA from challenging fish tissues
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TECHNICAL NOTE
Evaluation of three methods for high throughput extraction of DNA from challenging fish tissues Helge Meissner • Svein-Erik Fevolden Per-Arne Amundsen • Kim Præbel
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Received: 12 November 2012 / Accepted: 21 February 2013 / Published online: 1 March 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Three methods for extracting DNA were tested on otoliths, scales, fins, and gill tissue from European whitefish [Coregonus lavaratus (L.)]. The aim was to find time-efficient and affordable ways to simultaneously extract DNA suitable for conservation genetic studies from a large number of samples and different tissues. A rapid low-cost method led to 97 % success of microsatellite amplification in otoliths and 100 % in scales. High amplification success was achieved with fin (97 %) and gill (99 %) tissue using a salt lysis-based protocol. A commercial extraction kit delivered good results with all tissues. The findings are useful for conservation genetic studies using both contemporary and archived samples. Keywords Plate extraction of DNA Salmonids Population genetics Non-lethal sampling
Rationalizing DNA extraction from fish has been the focus in several recent studies (Wasko et al. 2003; Chakraborty et al. 2006; Lucentini et al. 2006; Nielsen and Hansen 2008; Campbell and Narum 2009). However, a comparison of high through-put DNA extraction methods useful in fish species conservation studies, which typically employ nonlethal sampling (scales, fin clips) or samples of low DNA content (scales, otoliths), has yet to be performed. Here, we H. Meissner S.-E. Fevolden P.-A. Amundsen K. Præbel (&) Department of Arctic and Marine Biology, University of Tromsø, 9037 Tromsø, Norway e-mail: [email protected] Present Address: H. Meissner ˚ s, Norway Norwegian Forest and Landscape Institute, 1431 A
test one method from each of the three main principles used for fish DNA extraction for genotyping studies; binding DNA to a membrane matrix, salt/solvent precipitation of DNA, and osmotic bursting of cells to release DNA. The aim was to identify an accurate and consistent, yet time/ money-efficient high through-put extraction method for each type of tissue. Ten European whitefish [Coregonus lavaratus (L.)] stored frozen for 1 year at -20 °C were sampled for sagittal otoliths, scales, gill and adipose fin tissue. Scales were stored in paper bags at room temperature for 1 month, whereas all other materials were preserved in 96 % ethanol. Gills, fins, and scales were analyzed in triplicate for each fish. For all extractions, a piece of fin or gill tissue (about 5 mg in weight), one complete scale (about 0.5 mg), or one half of a sagittal otolith (about 10 mg) were subjected to each of the following treatments: The Qiagen DNeasy kit, representing the matrix-based DNA extraction methods, was used following the manufacturer’s instructions. DNA yield was increased by performing the final elution step twice using 50 or 25 ll elution buffer for gills/fin tissue or otoliths/scales, respectively. A prot
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