This article details a preprocessing method for extracting genomic DNA from shed reptile skin using the Wizard® SV Genomic Purification System (Cat.# A2360). The advantage to using shed skin is that genetic studies of herpetofauna can be conducted without taking potentially harmful samples of blood or other tissues. The shed skin extraction method has been developed as part of an ongoing molecular analysis of the captive population of bearded dragons (Pogona vitticeps) to understand their geographic origin. This study requires the use of shed skin samples because concerned pet owners are unable or unwilling to provide the types of tissue from which genomic DNA is typically extracted (e.g., blood, liver or muscle). Use of this method offers a noninvasive approach to collecting DNA samples that allows a broader opportunity to study reptile populations.
Figure 1. Image of a bearded dragon (Pogona vitticeps
Image courtesy of mrskingsbioweb.com
Material and Methods
Shed skin samples were obtained from reptile breeders and enthusiasts across the continental United States. Labeled collection tubes were sent with sample information sheets to each participant to maintain sample identity. Each breeder was asked to record the age, size, sex and morph (color and pattern) of each individual on these data sheets. Photo vouchers were also requested to keep records of the physical appearance of each reptile. Participants were instructed to obtain 2 square inches of reptile shed from each specimen, and to allow the skin sample to dry completely before shipping to avoid contamination. Samples were approximately the same age prior to processing, with a variance of 2–3 weeks. This protocol was successfully tested on 13 skin samples and one liver sample from three species of lizard.
Preprocessing of the Reptile Shed Skin
- Prepare enough Digestion Solution for the number of samples to be isolated (see table below).
- Cut one square inch of shed skin sample into quarters.
- Place the four quarters into 1.5ml tube containing Digestion Solution.
- Attach disposable plastic pestle to electric drill.
(Note: It is important to have a pestle and tube that match one another to ensure proper mastication of the skin sample.)
- Thoroughly grind skin sample in Digestion Solution until sample becomes a slurry.
- Incubate sample for 48 hours in a 55°C heat block or water bath, vortexing the sample periodically.
- Centrifuge sample at 6,500 × g for 2 minutes.
- Transfer supernatant to a new labeled 1.5ml tube with a 200µl pipette tip, leaving the cell debris undisturbed.
- Repeat Steps 7 and 8.
- Add 500µl of Wizard® SV Lysis Buffer to each sample, and vortex to mix.
- Process samples using the Vac-Man® Vacuum Manifold as detailed in the Purification of Genomic DNA from Lysates Using a Vac-Man® Vacuum Manifold section of the Wizard® SV Genomic DNA Purification System Technical Bulletin #TB302. For some samples, small amounts of cell debris remained and the centrifugation purification method in the Purification of Genomic DNA from Lysates Using a Microcentrifuge section was used to avoid minicolumn clogging.
Table 1. Digestion Solution Preparation.
To confirm the success of DNA extraction, temperature gradient PCR was used to amplify a ~1,200 bp fragment of mitochondrial DNA (mtDNA) centered around the NADH dehydrogenase 2 (ND2) gene using available primers. See Table 2 for reaction volumes and cycling conditions respectively. The temperature gradient PCR was performed in a single thermal cycler that can amplify samples with a range of annealing temperatures (Table 2). Gel electrophoresis was used to determine if the correctly sized PCR product was successfully amplified. Successfully amplified PCR products were then cleaned up and sequenced to confirm their identity.
Table 2. PCR Mix and Cycling Conditions.
After testing digestion times of 16–48 hours, the first successful extraction was obtained using a Leopard Gecko (Eublepharis macularius) shed skin sample digested for 48 hours. Amplification of the purified DNA produced a bright distinct band of ~1,200 bp after gel electrophoresis and SYBR® staining of the PCR product. Attempts to extract DNA were made from a total of 29 samples. Of these, the expected PCR product was obtained from 13 skin samples of P. vitticeps, even though the shed samples were much harder to masticate than that of E. macularius, and one liver sample from Anolis cristatellus, which was used as the positive control for the modified protocol. Successful sequencing of the mitochondrial ND2 region was obtained from 10 of the 14 samples. Figure 2 shows gradient PCR results of three samples with annealing temperatures 51.5–68°C. DNA quantification (absorbance at 260 nm) was performed on all eluted samples. The liver sample from A. cristatellus had a DNA content of 18.2ng/µl. Of the skin samples extracted, DNA yield ranged from 4.2ng/µl to 95.9ng/µl, with a mean of 18ng/µl.
Figure 2. Annealing temperature gradient reactions performed for three Pogona
Using the protocol outlined in the Methods section, annealing temperature gradient PCR was performed for three Pogona skin samples. For each of eight different annealing temperatures, 2µl samples mixed with 6X bromophenol blue loading dye were separated on a 2% agarose gel containing 5µl of SYBR® Safe DNA Gel Stain (Invitrogen). The prominent band represents the expected 1,200 base pair product. Lane 1, 51.5°C; lane 2, 53.4°C; lane 3, 55.7°C; lane 4, 58.3°C; lane 5, 61°C; lane 6, 63.7°C; lane 7, 66.1°C; lane 8, 68°C; lane 9, 2µl of Quick-Load® 2-Log DNA Ladder (0.1–10kb; New England Biolabs).
Shed skin offers a viable nondestructive alternative to tissue, blood or other DNA sources and provides high-quality DNA for study. In addition to its value for this specific application, DNA extraction from shed skin may also permit analysis of samples obtained from the field. If the skin sample can be identified at the species level (as is often the case for shed snake skins in the United States), these specimens may be used to supplement sampling for standard molecular population genetic or phylogenetic analyses. If the shed skin cannot be identified based on its physical appearance, this protocol may permit its identification on the basis of molecular markers. In this sense, this protocol may be a useful tool for herpetological surveys. This modified protocol broadens the opportunity for sampling of populations and decreases the effect of study on those populations.