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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ftpubmed:oai:pubmedcentral.nih.gov:7147431 2023-05-15T18:42:26+02:00 A real-time PCR assay to accurately quantify polar bear DNA in fecal extracts Hayward, Kristen M. Harwood, Michelle P. Lougheed, Stephen C. Sun, Zhengxin Van Coeverden de Groot, Peter Jensen, Evelyn L. 2020-04-07 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147431/ http://www.ncbi.nlm.nih.gov/pubmed/32292653 https://doi.org/10.7717/peerj.8884 en eng PeerJ Inc. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147431/ http://www.ncbi.nlm.nih.gov/pubmed/32292653 http://dx.doi.org/10.7717/peerj.8884 © 2020 Hayward et al. https://creativecommons.org/licenses/by-nc/4.0 This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by-nc/4.0) , which permits using, remixing, and building upon the work non-commercially, as long as it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. CC-BY-NC Biochemistry Text 2020 ftpubmed https://doi.org/10.7717/peerj.8884 2020-04-19T00:33:29Z 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. Text Ursus maritimus PubMed Central (PMC) PeerJ 8 e8884 |
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English |
topic |
Biochemistry |
spellingShingle |
Biochemistry Hayward, Kristen M. Harwood, Michelle P. Lougheed, Stephen C. Sun, Zhengxin Van Coeverden de Groot, Peter Jensen, Evelyn L. A real-time PCR assay to accurately quantify polar bear DNA in fecal extracts |
topic_facet |
Biochemistry |
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 |
Text |
author |
Hayward, Kristen M. Harwood, Michelle P. Lougheed, Stephen C. Sun, Zhengxin Van Coeverden de Groot, Peter Jensen, Evelyn L. |
author_facet |
Hayward, Kristen M. Harwood, Michelle P. Lougheed, Stephen C. Sun, Zhengxin Van Coeverden de Groot, Peter Jensen, Evelyn L. |
author_sort |
Hayward, Kristen M. |
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 |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147431/ http://www.ncbi.nlm.nih.gov/pubmed/32292653 https://doi.org/10.7717/peerj.8884 |
genre |
Ursus maritimus |
genre_facet |
Ursus maritimus |
op_relation |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147431/ http://www.ncbi.nlm.nih.gov/pubmed/32292653 http://dx.doi.org/10.7717/peerj.8884 |
op_rights |
© 2020 Hayward et al. https://creativecommons.org/licenses/by-nc/4.0 This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by-nc/4.0) , which permits using, remixing, and building upon the work non-commercially, as long as it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. |
op_rightsnorm |
CC-BY-NC |
op_doi |
https://doi.org/10.7717/peerj.8884 |
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8 |
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e8884 |
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1766232082041798656 |