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Michael Krützen, Ph.D. University of Zurich Anthropological Institute and Museum Winterthurerstr. 190 CH-8057 Zurich Switzerland Email: michael.kruetzen*aim.uzh.ch |
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Benoit Goossens, Ph.D Cardiff University PO Box 915 Cathays Park Cardiff CF10 3TL United Kingdom Email: goossensbr*cardiff.ac.uk |
Genetic analyses have become an extremely valuable tool to study multiple aspects of primate evolution. Sources of DNA usually vary between organisms, but usually researchers prefer to obtain DNA samples through non-invasive means, such as fecal material or shed hair. Several studies have shown that storage methods could potentially have a great effect on DNA quality and yield. Here, we describe four storage methods for faecal DNA that has been used by various research groups. We do not intend this list to be all inclusive, but rather focus on methods that allow storage of DNA material in difficult field conditions.
a) Availability of reagents and storage vials in the field
b) Storage conditions in the field
c) Transport of samples from field to laboratory
d) Cost of reagents
We have identified five different protocols. The first three are suited for fecal material, while the forth one works well with small pieces of tissue, if for instance a dead animal is encountered. The last protocol describes the storage and preservation of shed hair.
Fecal material should be collected as soon as possible after defecation. As a rule of thumb, all samples should be stored as cool as possible (preferably frozen, but (4°C works well, too). Unfortunately, in many field situations this is not possible. A possible solution is a so-called “dry-shipper” (sometimes called a “vapor-shipper”). These are fabricated from durable, lightweight aluminium, containing a hydrophobic absorbent that traps the liquid nitrogen. If properly filled, a dry shipper can cool specimens to -150°C for three weeks, even at high ambient temperatures. Dry-shippers are also approved my most airlines. If refrigerated storage is not possible in the field, the very least that should be done is storing the specimens at a cool and dry place. In areas of high humidity, this could be achieved by placing the samples in a sealable plastic container that contains silica gel to absorb the moisture. Generally, samples should be transferred to a lab as soon as possible.
IMPORTANT: Most of the shed cells of the target organism are found at the ‘front end’ and the outside of the fecal material. Hence, it is important to scrape as much as possible from the outside of the material in order to maximize DNA yield. To avoid contamination with human DNA, gloves should be worn at all times when handling samples (although we know that this is sometimes difficult to do in tropical environment). For each sample, a new pair of gloves should be used to minimize the risk of cross-contamination.
Materials required:
If yes: Monitor silica gel every two-three weeks for change in color, replace silica gel ONLY if necessary
If not dry: replace silica gel, check again after two to three days.
RNA later (manufactured by several companies, e.g. Ambion, Sigma-Aldrich, Qiagen and others!) is a specialized storage solution that was originally designed to preserve RNA in samples collected in the field. It appears to preserve DNA very well, and seems to have a superior performance compared to methods 1 and 2. Unfortunately, it is not cheap.
This method works very well to preserve small quantities of tissue at room temperature. It is basically like pickling meat. The DMSO helps to rupture cell structures, so the salt can penetrate the cells much quicker to denature proteins.
Goossens B, Anthony N, Jeffery K, Johnson-Bawe M and Bruford MW (2003). Collection, storage and analysis of non-invasive genetic material in primate biology. In Setchell JM, Curtis DC, ed. Field and Laboratory Methods in Primatology, pp. 295-308. Cambridge University Press, Cambridge.
Jeffery KJ, Abernethy KA, Tutin CEG, Bruford MW (2006) Biological and environmental degradation of Gorilla hair and microsatellite amplification success in hair extracted DNA. Biological Journal of the Linnean Society, in press.
Murphy MA, Waits LP, Kendall KC (2000) Quantitative evaluation of fecal drying methods for brown bear DNA analysis. Wildlife Society Bulletin, 28, 951-957.
Murphy MA, Waits LP, Kendall KC, Wasser SK, Higbee JA, Bogden R (2002) An evaluation of long-term preservation methods for brown bear (Ursus arctos) faecal DNA samples. Conservation Genetics, 3, 435-440.
Nsubuga AM, Robbins MM, Roeder AD, Morin PA, Boesch C, Vigilant L (2004) Factors affecting the amount of genomic DNA extracted from ape faeces and the identification of an improved sample storage method. Molecular Ecology, 13, 2089-2094.
Roeder AD, Archer FI, Poinar HN, Morin PA (2004) A novel method for collection and preservation of faeces for genetic studies. Molecular Ecology Notes, 4, 761-764.
Valderrama X, Karesh WB, Wildman DE, Melnick DJ (1999) Non-invasive methods for collecting fresh hair tissue. Molecular Ecology, 8, 1749-1752.
