Aurintricarboxylic acid increases yield of HSV-1 vectors

Pechan, Peter; Ardinger, Jeffery; Ketavarapu, Jyothi; Rubin, Hillard; Wadsworth, Samuel C; Scaria, Abraham

ed in the presence of 250 U/ml of Benzonase Endonuclease (EMD Millipore, Billerica, MA) in 2 mmol/l MgCl 2 , 1% final concentration protein grade Tween 80, and incubated at 37 °C for 60 minutes, followed by 0.25% Gibco Trypsin (Invitrogen Life Technologies) digestion at 50 °C for 60 minutes. Finally, treatment with DNase I (50 U/ ml final concentration) at 37 °C for 30 minutes was performed and then denaturation at 95 °C for 20 minutes. Linearized plasmid pDC67/+SV40 (Genzyme, Framingham, MA) was used to generate standard curves. The primer-probe set was specific for the simian virus 40 (SV40) poly (A) sequence: rAAV-F: 5′-AGCAATAGCATCACAAATTTCACAA-3′, rAAV-R: 5′-GCAGACATGATAAGATACATTGATGAGTT-3′, and rAAV-Probe: 5′ 6-FAM-AGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTC-TAMRA-3′. Amplification of the PCR product was achieved with the following cycling parameters: 1 cycle at 50 °C for 2 minutes, 1 cycle at 95 °C for 10 minutes; 40 cycles at 95 °C for 15 seconds, and 40 cycles at 60 °C for 60 seconds. The results were expressed as a mean of rAAV DRP/ml titers ± SD and were statistically analyzed by Prism 5.0d GraphPad Software.