Gutenberg Open Science: Transgene detection by digital droplet PCR

Please use this identifier to cite or link to this item: http://doi.org/10.25358/openscience-7583

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DC Field	Value	Language
dc.contributor.author	Moser, Dirk A.	-
dc.contributor.author	Braga, Luca	-
dc.contributor.author	Raso, Andrea	-
dc.contributor.author	Zacchigna, Serena	-
dc.contributor.author	Giacca, Mauro	-
dc.contributor.author	Simon, Perikles	-
dc.date.accessioned	2022-08-19T10:19:35Z	-
dc.date.available	2022-08-19T10:19:35Z	-
dc.date.issued	2014
dc.identifier.uri	https://openscience.ub.uni-mainz.de/handle/20.500.12030/7597	-
dc.description.abstract	Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.	en_GB
dc.description.sponsorship	DFG, Open Access-Publizieren Universität Mainz / Universitätsmedizin	de
dc.language.iso	eng	de
dc.rights	CC BY	*
dc.rights.uri	https://creativecommons.org/licenses/by/4.0/	*
dc.subject.ddc	796 Sport	de_DE
dc.subject.ddc	796 Athletic and outdoor sports and games	en_GB
dc.title	Transgene detection by digital droplet PCR	en_GB
dc.type	Zeitschriftenaufsatz	de
dc.identifier.doi	http://doi.org/10.25358/openscience-7583	-
jgu.type.dinitype	article	en_GB
jgu.type.version	Published version	de
jgu.type.resource	Text	de
jgu.organisation.department	FB 02 Sozialwiss., Medien u. Sport	de
jgu.organisation.number	7910	-
jgu.organisation.name	Johannes Gutenberg-Universität Mainz	-
jgu.rights.accessrights	openAccess	-
jgu.journal.title	PLoS one	de
jgu.journal.volume	9	de
jgu.journal.issue	11	de
jgu.pages.alternative	e111781	de
jgu.publisher.year	2014	-
jgu.publisher.name	PLoS	de
jgu.publisher.place	Lawrence, Kan.	de
jgu.publisher.uri	http://dx.doi.org/10.1371/journal.pone.0111781	de
jgu.publisher.issn	1932-6203	de
jgu.organisation.place	Mainz	-
jgu.identifier.pmid	25375130
jgu.subject.ddccode	796	de
opus.date.modified	2018-08-09T08:18:35Z
opus.subject.dfgcode	04-205
opus.organisation.string	FB 02: Sozialwissenschaften, Medien und Sport: Institut für Sportwissenschaft	de_DE
opus.identifier.opusid	50411
opus.importsource	pubmed
opus.institute.number	0208
opus.metadataonly	false
opus.type.contenttype	Keine	de_DE
opus.type.contenttype	None	en_EN
opus.affiliated	Simon, Perikles
jgu.publisher.doi	10.1371/journal.pone.0111781	de
jgu.organisation.ror	https://ror.org/023b0x485	-
Appears in collections:	DFG-OA-Publizieren (2012 - 2017)

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