Specific detection of RT activity in culture supernantants of retrovirus-producing cells, using synthetic DNA as competitor in polymerase enhanced reverse transcriptase assay.
The polymerase enhanced reverse transcriptase (PERT) assay is a highly sensitive assay for the detection of reverse transcriptase (RT) activity in culture supernatants of retrovirus-producing cells. However, some cellular DNA-dependent DNA polymerases exhibit RT-like activities in this assay. A synthetic DNA competitor which suppresses the RT-like activities of cellular DNA-dependent DNA polymerases was used in a modified PERT assay technique for specific detection of RT activity in culture supernatants of retrovirus-producing cells. We determined the optimum condition of the assay and evaluated its specificity. This improved PERT assay is easy to perform and is able to detect minute amounts of purified RT, as well as RT in crude cell lysates and concentrated culture supernatants.
Voisset
C
C
Unité Mixte de Recherche 2142 CNRS-bioMérieux, Ecole Normale Supérieure de Lyon, 46, allée d'Italie, 69364 Cedex 07, Lyon, France.
Tönjes
R R
RR
Breyton
P
P
Mandrand
B
B
Paranhos-Baccalà
G
G
D000818:Animals; D003470:Culture Media; D004247:DNA; D009052:Leukemia Virus, Murine / Q000201:enzymology; D051379:Mice; D011089:Polydeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D012194:RNA-Directed DNA Polymerase / Q000032:analysis* / Q000235:genetics; D012190:Retroviridae; D012680:Sensitivity and Specificity
D003470:Culture Media; D011089:Polydeoxyribonucleotides; C084838:polydeoxyadenylic acid-polythymidylic acid; D004247:DNA; D012194:RNA-Directed DNA Polymerase