Limits...
Transgene detection by digital droplet PCR.

Moser DA, Braga L, Raso A, Zacchigna S, Giacca M, Simon P - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Psychology, Genetic Psychology, Ruhr-University-Bochum, Bochum, Germany; Department of Sports Medicine, Disease Prevention and Rehabilitation, Johannes Gutenberg-University Mainz, Mainz, Germany.

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.

Show MeSH

Related in: MedlinePlus

DdPCR results following 3 different DNA extraction procedures.Blood of 5 donors was spiked with the same amount of circular plasmid containing the IGF1 coding sequence. DNA was extracted using 3 different procedures: uncut, DdeI and RsaI cut, and on-column DdeI and RsaI digested (ocd). Results are displayed as copies detected per µl for 5 subjects (I–V for uncut; conventionally digested (cut), on-column digested DNA (ocd), and no-template control (ntc). Results derive from two independent experiments performed in duplicates.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4222945&req=5

pone-0111781-g003: DdPCR results following 3 different DNA extraction procedures.Blood of 5 donors was spiked with the same amount of circular plasmid containing the IGF1 coding sequence. DNA was extracted using 3 different procedures: uncut, DdeI and RsaI cut, and on-column DdeI and RsaI digested (ocd). Results are displayed as copies detected per µl for 5 subjects (I–V for uncut; conventionally digested (cut), on-column digested DNA (ocd), and no-template control (ntc). Results derive from two independent experiments performed in duplicates.

Mentions: In addition, testing DNA extracted from whole blood (a- conventionally extracted; b- conventionally extracted followed by DNA digestion; c- on-column digested) that was spiked with the same amount of plasmid standard by ddPCR revealed more sensitive detection for digested DNA, and most sensitive detection for samples subjected to on-column digestion. As indicated in Figure 3, we could achieve 1.9–2.8 fold increased ddPCR sensitivity comparing digested DNA to undigested DNA. When ddPCR was performed from the same blood after on-column digestion, ddPCR performance was further increased 2.9–19 fold compared to DNA eluted using water (Figure 3).


Transgene detection by digital droplet PCR.

Moser DA, Braga L, Raso A, Zacchigna S, Giacca M, Simon P - PLoS ONE (2014)

DdPCR results following 3 different DNA extraction procedures.Blood of 5 donors was spiked with the same amount of circular plasmid containing the IGF1 coding sequence. DNA was extracted using 3 different procedures: uncut, DdeI and RsaI cut, and on-column DdeI and RsaI digested (ocd). Results are displayed as copies detected per µl for 5 subjects (I–V for uncut; conventionally digested (cut), on-column digested DNA (ocd), and no-template control (ntc). Results derive from two independent experiments performed in duplicates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222945&req=5

pone-0111781-g003: DdPCR results following 3 different DNA extraction procedures.Blood of 5 donors was spiked with the same amount of circular plasmid containing the IGF1 coding sequence. DNA was extracted using 3 different procedures: uncut, DdeI and RsaI cut, and on-column DdeI and RsaI digested (ocd). Results are displayed as copies detected per µl for 5 subjects (I–V for uncut; conventionally digested (cut), on-column digested DNA (ocd), and no-template control (ntc). Results derive from two independent experiments performed in duplicates.
Mentions: In addition, testing DNA extracted from whole blood (a- conventionally extracted; b- conventionally extracted followed by DNA digestion; c- on-column digested) that was spiked with the same amount of plasmid standard by ddPCR revealed more sensitive detection for digested DNA, and most sensitive detection for samples subjected to on-column digestion. As indicated in Figure 3, we could achieve 1.9–2.8 fold increased ddPCR sensitivity comparing digested DNA to undigested DNA. When ddPCR was performed from the same blood after on-column digestion, ddPCR performance was further increased 2.9–19 fold compared to DNA eluted using water (Figure 3).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Psychology, Genetic Psychology, Ruhr-University-Bochum, Bochum, Germany; Department of Sports Medicine, Disease Prevention and Rehabilitation, Johannes Gutenberg-University Mainz, Mainz, Germany.

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.

Show MeSH
Related in: MedlinePlus