High efficiency protocol of DNA extraction fromMicromys minutusmandibles from owl pellets: a tool for molecular research of cryptic mammal species.
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 mouseMicromys 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.
Museum and Institute of Zoology, Polish Academy of Sciences, Wilcza 64, 00679 Warsaw, Poland ; Department of Immunology, Genetics and Pathology, BMC, Uppsala University, Husargatan 3, 75108 Uppsala, Sweden.
Magdalena M
MM
Buś
Museum and Institute of Zoology, Polish Academy of Sciences, Wilcza 64, 00679 Warsaw, Poland.
Michał
M
Zmihorski
Centre for Ecological Research, Polish Academy of Sciences, Konopnickiej 1, 05092 Łomianki, Poland.
Jerzy
J
Romanowski
Nature Research Centre, Akademijos 2, 08412 Vilnius, Lithuania.
Laima
L
Balčiauskienė
Department of Zoology, University of Zielona Góra, Szafrana 1, 65516 Zielona Góra, Poland.
Jan
J
Cichocki
Nature Research Centre, Akademijos 2, 08412 Vilnius, Lithuania.
Linas
L
Balčiauskas
Amplification success rate; Bones; Degradation; Harvest mouse; Inhibition; Noninvasive genetic sampling