Improving DNA quality extracted from fecal samples—a method to improve DNA yield

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• 作者：Vânia Costa ; Sónia Rosenbom ; Rita Monteiro…
• 关键词：Fecal samples ; Equids ; DNA extraction ; Noninvasive sampling ; Protocol ; Next ; generation sequencing
• 刊名：European Journal of Wildlife Research
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：63
• 期：1
• 全文大小：
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Zoology; Ecology; Fish & Wildlife Biology & Management;
• 出版者：Springer Berlin Heidelberg
• ISSN：1439-0574
• 卷排序：63

文摘

The study and conservation of endangered species is an important topic but collecting genetic samples is challenging as many of the threatened species are rare, elusive, or difficult to approach. Thus, the use of noninvasive samples to obtain genetic information has been gaining popularity. The noninvasive samples generally yield low DNA quantity and quality, which interferes with the genetic analyses performed. Several studies have identified factors that influence the amount and integrity of DNA extracted from noninvasive samples. Our work introduces a noninvasive DNA extraction method that can be modified and adapted to recover a large amount of DNA from stool samples. The protocol described here consists of two digestions and two purification steps and was tested on samples collected in the field from wild animals. This new method proved to be effective in the DNA extraction from scat samples in different conditions and from different species. The concentration of extracted DNA ranged from 30 to 70 ng/μl for Equus hemionus, Equus africanus, and Equus kiang. The success of mtDNA amplification ranged between 80 and 100% while for microsatellite markers the rate was between 65 and 100%. The reproducibility between scorings of the amplified fragments ranged between 76 and 100%. The current method has successfully improved the DNA yield on several species of wild equids. In addition to the classic genetic markers, we also test this method suitability for next-generation sequencing methods, by obtaining a RADseq library of 16 nM using 16 cycles and fragment size distribution similar to what is expected for high-quality samples. This protocol has successfully improved the DNA yield on different species by changing some steps of a classical spin-column DNA extraction protocol. We conclude that our fecal DNA extraction method can be used to provide DNA for a variety of downstream analyses.