A real-time PCR assay to accurately quantify polar bear DNA in fecal extracts
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...
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ftdoajarticles:oai:doaj.org/article:882890d4232b4f0197d9c41342c9effa 2024-01-07T09:47:12+01:00 A real-time PCR assay to accurately quantify polar bear DNA in fecal extracts Kristen M. Hayward Michelle P. Harwood Stephen C. Lougheed Zhengxin Sun Peter Van Coeverden de Groot Evelyn L. Jensen 2020-04-01T00:00:00Z https://doi.org/10.7717/peerj.8884 https://doaj.org/article/882890d4232b4f0197d9c41342c9effa EN eng PeerJ Inc. https://peerj.com/articles/8884.pdf https://peerj.com/articles/8884/ https://doaj.org/toc/2167-8359 doi:10.7717/peerj.8884 2167-8359 https://doaj.org/article/882890d4232b4f0197d9c41342c9effa PeerJ, Vol 8, p e8884 (2020) qPCR Population genetics Noninvasive sampling Ursus maritimus Medicine R Biology (General) QH301-705.5 article 2020 ftdoajarticles https://doi.org/10.7717/peerj.8884 2023-12-10T01:51:08Z 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 F2 gene and a quantitative PCR (qPCR) assay that allows the accurate quantification of the amount of polar bear (Ursus maritimus) 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 F2 gene have been identified across vertebrates; thus, similar qPCR assays could be developed for other species to enable noninvasive studies. Article in Journal/Newspaper Ursus maritimus Directory of Open Access Journals: DOAJ Articles PeerJ 8 e8884 |
institution |
Open Polar |
collection |
Directory of Open Access Journals: DOAJ Articles |
op_collection_id |
ftdoajarticles |
language |
English |
topic |
qPCR Population genetics Noninvasive sampling Ursus maritimus Medicine R Biology (General) QH301-705.5 |
spellingShingle |
qPCR Population genetics Noninvasive sampling Ursus maritimus Medicine R Biology (General) QH301-705.5 Kristen M. Hayward Michelle P. Harwood Stephen C. Lougheed Zhengxin Sun Peter Van Coeverden de Groot Evelyn L. Jensen A real-time PCR assay to accurately quantify polar bear DNA in fecal extracts |
topic_facet |
qPCR Population genetics Noninvasive sampling Ursus maritimus Medicine R Biology (General) QH301-705.5 |
description |
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 F2 gene and a quantitative PCR (qPCR) assay that allows the accurate quantification of the amount of polar bear (Ursus maritimus) 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 F2 gene have been identified across vertebrates; thus, similar qPCR assays could be developed for other species to enable noninvasive studies. |
format |
Article in Journal/Newspaper |
author |
Kristen M. Hayward Michelle P. Harwood Stephen C. Lougheed Zhengxin Sun Peter Van Coeverden de Groot Evelyn L. Jensen |
author_facet |
Kristen M. Hayward Michelle P. Harwood Stephen C. Lougheed Zhengxin Sun Peter Van Coeverden de Groot Evelyn L. Jensen |
author_sort |
Kristen M. Hayward |
title |
A real-time PCR assay to accurately quantify polar bear DNA in fecal extracts |
title_short |
A real-time PCR assay to accurately quantify polar bear DNA in fecal extracts |
title_full |
A real-time PCR assay to accurately quantify polar bear DNA in fecal extracts |
title_fullStr |
A real-time PCR assay to accurately quantify polar bear DNA in fecal extracts |
title_full_unstemmed |
A real-time PCR assay to accurately quantify polar bear DNA in fecal extracts |
title_sort |
real-time pcr assay to accurately quantify polar bear dna in fecal extracts |
publisher |
PeerJ Inc. |
publishDate |
2020 |
url |
https://doi.org/10.7717/peerj.8884 https://doaj.org/article/882890d4232b4f0197d9c41342c9effa |
genre |
Ursus maritimus |
genre_facet |
Ursus maritimus |
op_source |
PeerJ, Vol 8, p e8884 (2020) |
op_relation |
https://peerj.com/articles/8884.pdf https://peerj.com/articles/8884/ https://doaj.org/toc/2167-8359 doi:10.7717/peerj.8884 2167-8359 https://doaj.org/article/882890d4232b4f0197d9c41342c9effa |
op_doi |
https://doi.org/10.7717/peerj.8884 |
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PeerJ |
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8 |
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e8884 |
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1787429193099771904 |