A new DNA extraction method (HV-CTAB-PCI) for amplification of nuclear markers from open ocean-retrieved faeces of an herbivorous marine mammal, the dugong
Non-invasively collected faecal samples are an alternative source of DNA to tissue samples, that may be used in genetic studies of wildlife when direct sampling of animals is difficult. Although several faecal DNA extraction methods exist, their efficacy varies between species. Previous attempts to...
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| Veröffentlicht in: | PloS one 2023-06, Vol.18 (6), p.e0278792-e0278792 |
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| creator | Ooi, Vicky McMichael, Lee Hunter, Margaret E Takoukam Kamla, Aristide Lanyon, Janet M |
| description | Non-invasively collected faecal samples are an alternative source of DNA to tissue samples, that may be used in genetic studies of wildlife when direct sampling of animals is difficult. Although several faecal DNA extraction methods exist, their efficacy varies between species. Previous attempts to amplify mitochondrial DNA (mtDNA) markers from faeces of wild dugongs (Dugong dugon) have met with limited success and nuclear markers (microsatellites) have been unsuccessful. This study aimed to establish a tool for sampling both mtDNA and nuclear DNA (nDNA) from dugong faeces by modifying approaches used in studies of other large herbivores. First, a streamlined, cost-effective DNA extraction method that enabled the amplification of both mitochondrial and nuclear markers from large quantities of dugong faeces was developed. Faecal DNA extracted using a new 'High Volume- Cetyltrimethyl Ammonium Bromide- Phenol-Chloroform-Isoamyl Alcohol' (HV-CTAB-PCI) method was found to achieve comparable amplification results to extraction of DNA from dugong skin. As most prevailing practices advocate sampling from the outer surface of a stool to maximise capture of sloughed intestinal cells, this study compared amplification success of mtDNA between the outer and inner layers of faeces, but no difference in amplification was found. Assessment of the impacts of faecal age or degradation on extraction, however, demonstrated that fresher faeces with shorter duration of environmental (seawater) exposure amplified both markers better than eroded scats. Using the HV-CTAB-PCI method, nuclear markers were successfully amplified for the first time from dugong faeces. The successful amplification of single nucleotide polymorphism (SNP) markers represents a proof-of-concept showing that DNA from dugong faeces can potentially be utilised in population genetic studies. This novel DNA extraction protocol offers a new tool that will facilitate genetic studies of dugongs and other large and cryptic marine herbivores in remote locations. |
| doi_str_mv | 10.1371/journal.pone.0278792 |
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Although several faecal DNA extraction methods exist, their efficacy varies between species. Previous attempts to amplify mitochondrial DNA (mtDNA) markers from faeces of wild dugongs (Dugong dugon) have met with limited success and nuclear markers (microsatellites) have been unsuccessful. This study aimed to establish a tool for sampling both mtDNA and nuclear DNA (nDNA) from dugong faeces by modifying approaches used in studies of other large herbivores. First, a streamlined, cost-effective DNA extraction method that enabled the amplification of both mitochondrial and nuclear markers from large quantities of dugong faeces was developed. Faecal DNA extracted using a new 'High Volume- Cetyltrimethyl Ammonium Bromide- Phenol-Chloroform-Isoamyl Alcohol' (HV-CTAB-PCI) method was found to achieve comparable amplification results to extraction of DNA from dugong skin. As most prevailing practices advocate sampling from the outer surface of a stool to maximise capture of sloughed intestinal cells, this study compared amplification success of mtDNA between the outer and inner layers of faeces, but no difference in amplification was found. Assessment of the impacts of faecal age or degradation on extraction, however, demonstrated that fresher faeces with shorter duration of environmental (seawater) exposure amplified both markers better than eroded scats. Using the HV-CTAB-PCI method, nuclear markers were successfully amplified for the first time from dugong faeces. The successful amplification of single nucleotide polymorphism (SNP) markers represents a proof-of-concept showing that DNA from dugong faeces can potentially be utilised in population genetic studies. This novel DNA extraction protocol offers a new tool that will facilitate genetic studies of dugongs and other large and cryptic marine herbivores in remote locations.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0278792</identifier><identifier>PMID: 37285349</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Ammonium ; Ammonium bromides ; Amplification ; Analysis ; Animals ; Aquatic mammals ; Biology and Life Sciences ; Bromides ; Caniformia ; Cetacea ; Cetrimonium ; Cetyltrimethylammonium bromide ; Chemical analysis ; Chloroform ; Deoxyribonucleic acid ; DNA ; DNA, Mitochondrial - genetics ; DNA, Mitochondrial - metabolism ; Dugong ; Dugongs ; Earth Sciences ; Feces ; Genetic aspects ; Genomes ; Herbivores ; Herbivory ; Isopentyl alcohol ; Marine mammals ; Microsatellites ; Mitochondrial DNA ; Nucleotides ; Oceans and Seas ; Percutaneous Coronary Intervention ; Phenols ; Polymorphism ; Population genetics ; Population studies ; Research and analysis methods ; Sampling ; Seawater ; Single-nucleotide polymorphism ; Skin ; Success ; Water analysis ; Wildlife</subject><ispartof>PloS one, 2023-06, Vol.18 (6), p.e0278792-e0278792</ispartof><rights>Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. 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This novel DNA extraction protocol offers a new tool that will facilitate genetic studies of dugongs and other large and cryptic marine herbivores in remote locations.</description><subject>Ammonium</subject><subject>Ammonium bromides</subject><subject>Amplification</subject><subject>Analysis</subject><subject>Animals</subject><subject>Aquatic mammals</subject><subject>Biology and Life Sciences</subject><subject>Bromides</subject><subject>Caniformia</subject><subject>Cetacea</subject><subject>Cetrimonium</subject><subject>Cetyltrimethylammonium bromide</subject><subject>Chemical analysis</subject><subject>Chloroform</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA, Mitochondrial - genetics</subject><subject>DNA, Mitochondrial - metabolism</subject><subject>Dugong</subject><subject>Dugongs</subject><subject>Earth Sciences</subject><subject>Feces</subject><subject>Genetic 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new DNA extraction method (HV-CTAB-PCI) for amplification of nuclear markers from open ocean-retrieved faeces of an herbivorous marine mammal, the dugong</title><author>Ooi, Vicky ; McMichael, Lee ; Hunter, Margaret E ; Takoukam Kamla, Aristide ; Lanyon, Janet M</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c594t-acc27707f00709af4f6dcb296eb2c23a06551567fcf769e5397a5be0b41112f63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2023</creationdate><topic>Ammonium</topic><topic>Ammonium bromides</topic><topic>Amplification</topic><topic>Analysis</topic><topic>Animals</topic><topic>Aquatic mammals</topic><topic>Biology and Life Sciences</topic><topic>Bromides</topic><topic>Caniformia</topic><topic>Cetacea</topic><topic>Cetrimonium</topic><topic>Cetyltrimethylammonium bromide</topic><topic>Chemical analysis</topic><topic>Chloroform</topic><topic>Deoxyribonucleic 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Although several faecal DNA extraction methods exist, their efficacy varies between species. Previous attempts to amplify mitochondrial DNA (mtDNA) markers from faeces of wild dugongs (Dugong dugon) have met with limited success and nuclear markers (microsatellites) have been unsuccessful. This study aimed to establish a tool for sampling both mtDNA and nuclear DNA (nDNA) from dugong faeces by modifying approaches used in studies of other large herbivores. First, a streamlined, cost-effective DNA extraction method that enabled the amplification of both mitochondrial and nuclear markers from large quantities of dugong faeces was developed. Faecal DNA extracted using a new 'High Volume- Cetyltrimethyl Ammonium Bromide- Phenol-Chloroform-Isoamyl Alcohol' (HV-CTAB-PCI) method was found to achieve comparable amplification results to extraction of DNA from dugong skin. As most prevailing practices advocate sampling from the outer surface of a stool to maximise capture of sloughed intestinal cells, this study compared amplification success of mtDNA between the outer and inner layers of faeces, but no difference in amplification was found. Assessment of the impacts of faecal age or degradation on extraction, however, demonstrated that fresher faeces with shorter duration of environmental (seawater) exposure amplified both markers better than eroded scats. Using the HV-CTAB-PCI method, nuclear markers were successfully amplified for the first time from dugong faeces. The successful amplification of single nucleotide polymorphism (SNP) markers represents a proof-of-concept showing that DNA from dugong faeces can potentially be utilised in population genetic studies. This novel DNA extraction protocol offers a new tool that will facilitate genetic studies of dugongs and other large and cryptic marine herbivores in remote locations.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>37285349</pmid><doi>10.1371/journal.pone.0278792</doi><orcidid>https://orcid.org/0000-0002-9964-4038</orcidid><orcidid>https://orcid.org/0000-0002-7675-4859</orcidid><oa>free_for_read</oa></addata></record> |
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| recordid | cdi_plos_journals_2823477431 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Public Library of Science (PLoS) Journals Open Access; EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Ammonium Ammonium bromides Amplification Analysis Animals Aquatic mammals Biology and Life Sciences Bromides Caniformia Cetacea Cetrimonium Cetyltrimethylammonium bromide Chemical analysis Chloroform Deoxyribonucleic acid DNA DNA, Mitochondrial - genetics DNA, Mitochondrial - metabolism Dugong Dugongs Earth Sciences Feces Genetic aspects Genomes Herbivores Herbivory Isopentyl alcohol Marine mammals Microsatellites Mitochondrial DNA Nucleotides Oceans and Seas Percutaneous Coronary Intervention Phenols Polymorphism Population genetics Population studies Research and analysis methods Sampling Seawater Single-nucleotide polymorphism Skin Success Water analysis Wildlife |
| title | A new DNA extraction method (HV-CTAB-PCI) for amplification of nuclear markers from open ocean-retrieved faeces of an herbivorous marine mammal, the dugong |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-26T12%3A23%3A01IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=A%20new%20DNA%20extraction%20method%20(HV-CTAB-PCI)%20for%20amplification%20of%20nuclear%20markers%20from%20open%20ocean-retrieved%20faeces%20of%20an%20herbivorous%20marine%20mammal,%20the%20dugong&rft.jtitle=PloS%20one&rft.au=Ooi,%20Vicky&rft.date=2023-06-07&rft.volume=18&rft.issue=6&rft.spage=e0278792&rft.epage=e0278792&rft.pages=e0278792-e0278792&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0278792&rft_dat=%3Cgale_plos_%3EA752130958%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2823477431&rft_id=info:pmid/37285349&rft_galeid=A752130958&rft_doaj_id=oai_doaj_org_article_d6b4912bacac48129cc984299c08e96b&rfr_iscdi=true |