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Published on: October 5, 2017
DNA Extraction Optimisation for Minute Land Snails of Vertigo Müller, 1773 (Gastropoda: Vertiginidae): A Comparative
Benediktas Jukonis1, Jekaterina Havelka1, Grita Skujienė1
1Department of Zoology, Institute of Biosciences, Life Science Center Vilnius University Vilnius Lithuania.
Abstract:
No systematic comparison of DNA extraction strategies exists for minute Vertiginidae (shell height < 3 mm), a group posing a dual analytical challenge: extremely low tissue input and co-purified PCR-inhibitory mucus. For legally protected species, an additional requirement to preserve the shell voucher further constrains available protocols. Using Vertigo antivertigo as the model species, we compared six approaches applied to specimens preserved in 96% ethanol (n = 10 per method): two HotSHOT alkaline-lysis protocols (destructive and non-destructive shell-preserving variants), a modified CTAB protocol supplemented with PVP-40 and DTT, and three commercial silica-column kits (GeneJET Genomic, DNeasy Blood & Tissue, QIAamp DNA Micro). DNA yields were quantified by QuantiFluor fluorometry, and PCR performance was subsequently assessed across four loci (COI barcode, COI mini-barcode, ITS1, ITS2). DNeasy Blood & Tissue produced the highest fluorometric concentrations; QIAamp DNA Micro and CTAB + PVP-40 gave intermediate values. The shell-preserving HotSHOT variant yielded lower concentrations but improved A260/230 ratios. BSA and trehalose supplementation increased PCR success in inhibition-prone HotSHOT extracts from 70% to 100%. ITS1 Sanger sequencing of three Vertigo species listed in Annex II of the EU Habitats Directive, all extracted with the shell-preserving protocol, confirmed species-level identification (99.8%-100% BLASTn identity; mean Phred Q > 51). The shell-preserving non-destructive HotSHOT protocol yields sequenceable DNA from protected Vertiginidae while retaining the morphological voucher, making it the preferred option for conservation-genetic monitoring. The practical decision framework documented here-integrating voucher preservation, amplification robustness and per-sample cost-has broad applicability to other minute terrestrial gastropods processed in large-scale biodiversity surveys.
