A real-time PCR assay to accurately quantify polar bear DNA in fecal extracts

• Published in: PeerJ (San Francisco, CA) Vol. 8; p. e8884
• Main Authors: Hayward, Kristen M, Harwood, Michelle P, Lougheed, Stephen C, Sun, Zhengxin, Van Coeverden de Groot, Peter, Jensen, Evelyn L
• Format: Journal Article
• Language: English
• Published: United States PeerJ. Ltd 07-04-2020; PeerJ, Inc; PeerJ Inc
• Subjects: Animal behavior; Bacteria; Bears; Biochemistry; Deoxyribonucleic acid; DNA; DNA sequencing; Evolutionary Studies; Feces; Fishes; Genes; Genetic polymorphisms; Genetics; Genomes; Genotyping; Laboratories; Molecular Biology; Next-generation sequencing; Noninvasive sampling; Pathogenic microorganisms; Polar bear; Polymerase chain reaction; Population genetics; qPCR; Single nucleotide polymorphisms; Single-nucleotide polymorphism; Species; Time; Ursus maritimus; Wildlife; Zoology; Arctic; Canada; Nunavut Canada; United States > US; Arctic region; qPCR; Noninvasive sampling; Population genetics; Ursus maritimus
• ISSN: 2167-8359; 2167-8359
• DOI: 10.7717/peerj.8884

DNA extracted from fecal samples contains DNA from the focal species, food, bacteria and pathogens. Most DNA quantification methods measure total DNA and cannot differentiate among sources. Despite the desirability of noninvasive fecal sampling for studying wildlife populations, low amounts of focal species DNA make it difficult to use for next-generation sequencing (NGS), where accurate DNA quantification is critical for normalization. Two factors are required prior to using fecal samples in NGS libraries: (1) an accurate quantification method for the amount of target DNA and (2) a determination of the relative amount of target DNA needed for successful single nucleotide polymorphism genotyping assays. Here, we address these needs by developing primers to amplify a 101 bp region of the nuclear
gene and a quantitative PCR (qPCR) assay that allows the accurate quantification of the amount of polar bear (
) DNA in fecal extracts. We test the assay on pure polar bear DNA extracted from muscle tissue and find a high correlation between fluorometric and qPCR quantifications. The qPCR assay was also successfully used to quantify the amount of DNA derived from polar bears in fecal extractions. Orthologs of the
gene have been identified across vertebrates; thus, similar qPCR assays could be developed for other species to enable noninvasive studies.