Characterization of target nuclear DNA from faeces reduces technical issues associated with the assumptions of low-quality and quantity template

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• 作者：Mark C. Ball ; Richard Pither ; Micheline Manseau ; Jeff Clark ; Stephen D. Petersen ; Steve Kingston ; Natasha Morrill and Paul Wilson
• 关键词：Non ; invasive ; DNA quantification ; Population genetics ; Swift fox ; Woodland caribou
• 刊名：Conservation Genetics
• 出版年：2007
• 出版时间：June, 2007
• 年：2007
• 卷：8
• 期：3
• 页码：577-586
• 全文大小：264 KB

文摘

Faecal material has increasingly become an important non-invasive source of DNA for wildlife population genetics. However, DNA from faecal sources can have issues associated with quantity (low-template and/or low target-to-total DNA ratio) and quality (degradation and/or low DNA-to-inhibitor ratio). A number of studies utilizing faecal material assume and compensate for the above properties with minimal characterization of quantity or quality of target DNA, which can unnecessarily increase the risk of downstream technical problems. Here, we present a protocol which quantifies faecal DNA using a two step approach: (1) estimating total DNA concentration using a PicogreenTM fluorescence assay and (2) estimating target nuclear DNA concentration by comparing amplification products of field samples at suspected concentrations to those of control DNA at known concentrations. We applied this protocol to faecal material collected in the field from two species: woodland caribou (Rangifer tarandus) and swift fox (Vulpes velox). Total DNA estimates ranged from 6.5 ng/μl to 28.6 ng/μl (X = 16.2 ng/μl) for the caribou extracts and 1.0–26.1 ng/μl (X = 7.5 ng/μl) for the swift fox extracts. Our results showed high concordance between total and target DNA estimates from woodland caribou faecal extracts, with only 10 % of the samples showing relatively lower target-to-total DNA ratios. In contrast, DNA extracts from swift fox scat exhibited low target DNA yields, with only 38 % (19 of 50) of the samples showing comparative target DNA amplification of at least 0.1 ng. With this information, we were able to estimate the amount of target DNA entered into PCR amplifications, and identify samples having target DNA below a lower threshold of 0.2 ng and requiring modification to genotyping protocols such as multiple tube amplification. Our results here also show that this approach can easily be adapted to other species where faeces are the primary source of DNA template.