High efficiency protocol of DNA extraction from Micromys minutus mandibles from owl pellets: a tool for molecular research of cryptic mammal species.

Acta Theriologica Pub Date : 2014-01-01 Epub Date: 2013-05-10 DOI:10.1007/s13364-013-0144-y
Magdalena M Buś, Michał Zmihorski, Jerzy Romanowski, Laima Balčiauskienė, Jan Cichocki, Linas Balčiauskas
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引用次数: 1

Abstract

Owl pellets have high potential as a source of DNA. However, this noninvasive method of collecting DNA is rarely used, and its methodological aspects are poorly understood. We investigated the methodology for DNA extraction and amplification from owl pellets containing the smallest European rodent-the Harvest mouse Micromys minutus-as an example. We used mandibles identified in owl pellets for mitochondrial and nuclear DNA amplification. For DNA extraction, we tested two commercial protocols and utilized a protocol being a combination of two commercial kits which ensured high efficiency of DNA extraction. Additionally, we recorded that the amount of DNA was five times higher in extracts from teeth as compared to DNA extracts from jawbones derived from the same mandible. The quantity of DNA was significantly positively correlated with biological sample weight; however, the age of the pellet remains had an impact on the level of inhibition. We recorded inhibition in 40 % of mtDNA extracts derived from pellets older than 150 months, whereas in DNA extracts from pellets younger than 80 months, we did not observe a negative impact of inhibition on PCR efficiency. The amplification success rate was 89.9 % for the mitochondrial fragment and 39.4 % in the case of the nuclear fragment. We observed partial degradation of DNA evidenced by the fact that the longest fragments that we were able to amplify in the case of mtDNA were 450 and 200 bp for nuDNA. The study shows that pellets can be considered as a source of DNA and have high potential for molecular research in the case of threatened species and species that are difficult to study using standard field techniques.

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从猫头鹰颗粒中提取下颌骨DNA的高效方法:一种用于隐匿哺乳动物分子研究的工具。
猫头鹰丸作为DNA来源的潜力很大。然而,这种收集DNA的非侵入性方法很少被使用,其方法方面的理解也很差。我们研究了从含有最小的欧洲啮齿动物- - - Harvest鼠- - -的猫头鹰颗粒中提取和扩增DNA的方法。我们使用猫头鹰颗粒中鉴定的下颌骨进行线粒体和核DNA扩增。对于DNA提取,我们测试了两种商业协议,并使用了一种协议是两种商业试剂盒的组合,以确保DNA提取的高效率。此外,我们还记录到,从牙齿中提取的DNA含量是从同一下颌骨中提取的DNA含量的五倍。DNA含量与生物样品质量呈显著正相关;然而,颗粒残留物的年龄对抑制水平有影响。我们记录了来自超过150个月的颗粒的40%的mtDNA提取物的抑制作用,而来自小于80个月的颗粒的DNA提取物,我们没有观察到抑制对PCR效率的负面影响。线粒体片段的扩增成功率为89.9%,核片段的扩增成功率为39.4%。我们观察到DNA的部分降解,事实证明,我们能够在mtDNA的情况下扩增的最长片段是450和200 bp的nuDNA。该研究表明,颗粒可以被视为DNA的来源,并且在使用标准现场技术难以研究的濒危物种和物种的情况下具有很高的分子研究潜力。
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Acta Theriologica
Acta Theriologica 生物-动物学
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