I am working with a professor on a project to identify the phylogenetic affinity of rat snakes in the area, and we have been told that the DNeasy kit is good way to extract DNA. The samples we have are ethanol-preserved scales, but the official protocol does not address scales. I found an unofficial protocol that states how to use hair, nails, or feathers using this kit, which my research advisor believes will put us on the right track as these are all made of keratin. I have attached the adjusted protocol, but one of the professors who is helping me on this project wanted me to find out how the adjustments from the official protocol is supposed to work at the chemical level. I am having trouble finding any information about this.
Additionally, I am curious to know how many scales would be necessary in order to get a usable amount of DNA. From what I have read, the minimum appears to be about 10mg of a sample, but I would like some confirmation on this.
Hi Brenna,
I am not familiar at DNA extraction of reptiles. There is a paper (Vences et al., 2012) announces that it is possible to extract DNA from reptiles skins, such as scale clips and shed skin :
DNA Barcoding Amphibians and Reptiles
DOI: 10.1007/978-1-61779-591-6_5
I think you should only to expand the lysis time (maybe over 8 h) for the protein digestion.
About the second question, it just depend on your downstream experiment. If you want to implement PCR and sequencing, a small piece of scale will be enough.
Hi Brenna,
I have never worked with snake scales but, a few years ago, I was involved in a study working with feathers. We used the DNeasy Blood & Tissue Kit from Qiagen that worked pretty well.
All comments from your advisor and all modifications in the user-developed protocol are correct in my opinion. But I want to tell you our considerations for a successful DNA extraction from feathers.
Target material:
Similar to snake scales, our target material were epithelial cells adhering on feather tips.
DNA amount:
You never know how many cells do adhere there. This is an important issue: Unknown amount of starting biological material. In our case, we needed to identify bird individuals that we never saw. (They are protected species living in forests.) That means one feather = one individual. We never used more than one feather because we could not be sure that they would not belong to different individuals. This has nothing to do with the weight of the feather tip!
Quality of DNA:
Second issue is how "old" is the material? Our feathers were collected days, weeks or months after birds lost them. That means that DNA could be damaged to an unknown degree of degradation. In general, the fresher the material the better the DNA quality! Ethanol is a good way to store material protecting DNA from degradation.
In conclusion, we had an unknown amount of epithelial cells containing DNA of an unknown degradation level. Starting from a worst case scenario (= very few epithelial cells and highly degraded DNA), we performed lab work as follows:
1) Avoid cross-specimen contamination: We cut feather tips and put each in single tubes, never using the same scalpel and tweezers for different feathers. That means that after removing a feather tip, we destroyed presumable DNA residues on tools in 1.5% natriumhypochlorid solution during a few minutes, then washed twice in ddH2O. Then re-used tools.
2) Avoid contamination with PCR products: Preparation of feathers and DNA extraction was done in a pre-PCR room completely free of PCR products.
3) Incubation of feather tips in proteinase K (included in the DNAeasy kit) during a night at minimum (ca. 12h) to be sure that all epithelial cells were digested.
4) Then we followed the Qiagen protocol but - if you are using only 1 scale - I suggest to use less elution buffer. We used 2 x 75microL (instead of 2 x 100) to get a higher DNA concentration.
5) Finally, we used highly species-specific primers for PCR to avoid amplification due to cross-species contamination (since we did not know whether other animals touched these feathers or the collector contaminated them etc).
I hope these reflections can help you to perform a suitable protocol for your snake scales. Good luck!
Best regards
Carolina
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