Abstract
A recurring problem with ancient specimens from wildlife animals is that their preserved tissues contain small amounts of DNA in a degraded state. The specific objectives of the present study were: (1) To determine DNA yields from different animal tissues; (2) to compare traditional (manual) DNA extraction protocols with commercial procedures and (3) to assess the success of PCR amplification of Inter-Simple Sequence Repeats (ISSR) loci in degraded animal samples. Liver, stomach and muscle samples were extracted from coyote (Canis latrans) and long-tailed weasel (Mustela frenata) for this research. Manual protocols for DNA extraction were compared to a commercial kit procedure (Qiagen DNeasy kit). Genomic DNA in different states (intact, apoptotic and degraded) were amplified using a panel of ISSR primers. No DNA was recovered from coyote stomach samples using the manual extraction protocol. DNA concentrations in stomach and liver samples from coyote were 10.31 ng/μL and 15.8 ng/μL, respectively using the Qiagen extraction kit. In general, the kit extraction method yielded more DNA than the manual extraction procedure but it is more expensive. Intact and apoptotic genomic DNA were successfully amplified by PCR resulting in a similar profile. Artificially degraded DNA showed partial amplification. Thus, the ISSR marker system is suitable for animal population genetics when only limited and/or degraded animal DNA is available.
Highlights
Preserved tissues of ancient specimens of wildlife animals contain small amounts of DNA in a degraded state
All the genomic DNA samples were tested to assess their degradation level (Fig. 1 to 3). They were run in a 1% agarose gel with 0.5% Tris-Borate-EDTA (TBE) buffer
5 6 ng/μL of DNA were isolated from the same tissue using the Qiagen extraction kit
Summary
Preserved tissues of ancient specimens of wildlife animals contain small amounts of DNA in a degraded state. It is essential to make this DNA accessible for enzymatic manipulations. This normally involves the use of PCR amplification methods. Most DNA extraction protocols are suited for fresh tissues containing intact cells and high molecular weight DNA. To minimize DNA degradation, aggressive treatments including high temperatures and strong detergent should be avoided. These treatments may increase DNA release but would in turn decrease the overall DNA yield by further damaging the ancient DNA molecules (Rohland and Hofreiter, 2007)
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