From the frog's mouth: Buccal swabs for collection of DNA from amphibians

Abstract

Toe-clipping of amphibians for identification and DNA collection may have minimal impact on individuals in certain cases (Gelder and Strijbosch 1996) but has also been shown to result in decreased weight gain (Davis and Ovaska 2001), infection (Golay and Durrer 1994; Lemekert 1996), reduced breeding opportunities (Lemekert 1996), and decreased recapture rate indicating increased mortality (Clarke 1972). Genetic data are becoming important not only in determining the phylogeny of amphibian species, but also for examining population history (Bos and Sites 2001), hybridization (Arntzen 2001; Carpenter et al. 2001; Highton 1998), population structure (Newman and Squire 2001; Rowe et al. 2000, Shaffer et al. 2000), and estimating number of breeding adults in populations (Driscoll 1999; Scribner et al. 1997). These studies often involve multiple samples from sensitive species and populations; as amphibian populations around the world are declining (Houlahan et al. 2000), minimizing researcher impact to amphibian populations should be a priority. Buccal swabs have been used as an easy and non-invasive method of collecting tissue from humans for DNA extraction for many years (Thomson et al. 1992). We successfully used buccal swabs (WB10-0004; Whatman, Clifton, New Jersey, USA) to isolate workable amounts of high molecular weight DNA from barking frogs (Eleutherodactylus augusti). We brushed the interior of frog’s mouths for approximately 30 seconds per frog and immediately placed swabs in 650 μl of lysis buffer (50 mM Tris pH 8.0, 50 mM EDTA, 50 mM sucrose, 100 mM NaCl, 1% SDS). We stored swabs at room temperature overnight before refrigerating them at 4°C until extraction. We isolated DNA using a phenolchloroform extraction modified from Sambrook et al. (2001). Extractions began with the addition of 25 μl 10 mg/ml proteinase K followed by 55°C overnight incubation. After incubation, we washed this lysate with 650 μl phenol and then with 650 μl chloroform. We precipitated DNA by adding 0.1 volume 7.5 M sodium acetate and 0.6 volume isopropanol, followed by 0°C overnight incubation and subsequent centrifugation of samples for 30 minutes at 16,000 x g. We assessed the quality of DNA by running 4% of each sample in a 1% agarose gel and quantified using a microplate fluorescence reader (FLx800; Biotek Instruments, Inc., Winooski, Vermont, USA). From 33 swabs of barking frogs, we isolated an average of 1 μg high-quality DNA (95% CI 0.7 to 1.5 μg; 31.6 μg maximum) from each swab; we were unsuccessful in isolating DNA from only one swab. We also successfully extracted high molecular weight DNA from buccal swabs of bullfrogs (Rana catesbeiana), casque-headed frogs (Pternohyla fodiens), and lowland leopard frogs (Rana yavapaiensis). Researchers who wish to minimize disturbance to their study populations and whose research does not require over 1 μg of DNA should consider using buccal swabs as a low-impact alternative to toe-clipping or blood collection. Also, researchers who are storing tissue samples for future studies may wish to note that we extracted high molecular weight DNA from buccal swabs that had been in lysis buffer at room temperature for 2.5 years. Toe-clips preserved in ethanol for 1–4 years also yielded high molecular weight DNA, and other researchers have extracted high molecular weight DNA from tissue preserved in ethanol for up to 10 years (Li et al. 2000). However, one set of barking frog toe-clips preserved in ethanol away from light for 5 years yielded only degraded DNA.