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A quick one-tube nested PCR-protocol for EPO transgene detection
Drug testing and analysis, 2012-11, Vol.4 (11), p.870-875
Moser, Dirk A.
Neuberger, Elmo W. I.
Simon, Perikles
2012
Volltextzugriff (PDF)
Details
Autor(en) / Beteiligte
Moser, Dirk A.
Neuberger, Elmo W. I.
Simon, Perikles
Titel
A quick one-tube nested PCR-protocol for EPO transgene detection
Ist Teil von
Drug testing and analysis, 2012-11, Vol.4 (11), p.870-875
Ort / Verlag
England: Blackwell Publishing Ltd
Erscheinungsjahr
2012
Quelle
MEDLINE
Beschreibungen/Notizen
The practice of doping threatens fair competition in sports. With the very recent reports on successful gene therapies for several diseases, the likelihood for abuse of gene transfer techniques in elite sports is rapidly increasing. It is therefore very important to develop valid detection techniques for transgenic DNA (tDNA) with ultimate sensitivity and specificity. To date, three slightly different procedures have been reported to reliably detect tDNA with sufficiently high sensitivity. Two utilize a real‐time PCR‐based approach and one uses a primer‐internal, intron‐spanning PCR approach (spiPCR). The specificity and sensitivity of these techniques, however, is still a matter of debate. Based on spiPCR, here we present a novel one‐tube nested PCR approach that minimizes the chances for cross‐contamination and shows increased sensitivity compared to non‐nested PCR techniques. To further reduce the occurrence of false‐positives based on cross‐contamination, a multi‐functional 19bp extended erythropoietin standard (EPO) was cloned which can be easily differentiated from transgenic EPO DNA (tEPO) and can be used as an internal or external positive control in PCR‐based applications. We found that one‐tube nested PCR is superior in terms of sensitivity and specificity compared to conventional PCR, and shows similar sensitivity compared to real‐time based PCR assays. Although it did not reach sensitivity of spiPCR, the one‐tube nested PCR technique described here is less laborious, less expensive and much faster than spiPCR. This technique might therefore be useful as a pre‐screening tool for gene doping in the future. Copyright © 2012 John Wiley & Sons, Ltd. Doping is particularly difficult to combat when it involves pharmaceuticals and therapeutic techniques that are still development. Here, we present a novel one‐tube nested spiPCR approach that minimizes the chances for cross‐contamination and shows increased sensitivity compared to non‐nested PCR techniques. We hope that the sensitivity of the currently developed molecular biological detection techniques will deter athletes from using gene transfer doping techniques.
Sprache
Englisch
Identifikatoren
ISSN: 1942-7603
eISSN: 1942-7611
DOI: 10.1002/dta.1348
Titel-ID: cdi_proquest_miscellaneous_1197485871
Format
–
Schlagworte
antisense PCR
,
DNA - blood
,
DNA - genetics
,
DNA - isolation & purification
,
Doping in Sports
,
erythropoietin (EPO)
,
Erythropoietin - genetics
,
gene doping
,
Humans
,
one-tube nested PCR (OTN PCR)
,
Polymerase Chain Reaction - economics
,
Polymerase Chain Reaction - methods
,
Real-Time Polymerase Chain Reaction - economics
,
Real-Time Polymerase Chain Reaction - methods
,
SpiPCR
,
Time Factors
,
Transgenes
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