The HPLC running buffer was similar to the pH 7.5 buffer above but with 0.1 mg/mL H33258 and no su-Adenovirus is an icosahedral, nonenveloped, doublecrose. The flow rate for analysis was 0.5 mL/min. Five stranded DNA virus that promiscuously infects a broad to fifty microliters of dye-free sample was normally range of mammalian cell types, thereby rekindling inused and the analysis time per sample was 2 min. For terest in the use of recombinant adenovirus vectors for each sample, the area of the fluorescence peak was gene transfer into somatic cells both in vitro and in noted and to it was subtracted the value for a buffer vivo (1). Recombinant adenovirions are typically problank of the same volume used; the net fluorescence duced in a mammalian cell line; since only 10% of viral area was converted to amount of DNA from a standard DNA enters virus particles (2), viral DNA do accumucurve. All DNA values were reported as the average of late in amounts comparable to the total DNA of the triplicate measurements with the appropriate blank host cell. Subsequently, purification of the recombinant subtraction. material would require an effective removal of free vi-
For the experiments dealing with the effects of NaCl ral and host DNA for human therapeutics. To selecconcentration, pH, and temperature on adenovirus tively differentiate and quantitate free DNA from the samples, all sample manipulations were done at room DNA packaged inside the adenovirus capsid, we report temperature (22ΠC). The starting sample (1 mL; 2.9 1 in this study the development of a sensitive automated 10 11 particles) was in pH 7.5 buffer (see above) conmicrofluorometric procedure for DNA measurement ustaining 0.10 M NaCl with 0.1 mg/mL dye. For investigating the fluorescent dye Hoechst H33258 (3, 4) as a ing the effect of salt concentration, all samples were means of determining the quality as well as the stabildiluted twofold to the desired final NaCl concentrations ity of recombinant adenoviral samples. and were allowed to stand at room temperature for 30 Materials and methods. All reagents used were of min prior to DNA analysis. For the effect-of-pH experithe highest grade available. All manipulations were ments, the samples were adjusted to the desired pH as carried out at 4ΠC unless otherwise noted and under indicated in the table using 6 N HCl or 6 N NaOH and strict compliance with biological safety level 2 requirewere allowed to stand at room temperature for 30 min. ments. Recombinant Ad5 (rAd5) containing the p53
Samples with pH's other than 7.5 were then titrated gene was produced and purified as described previously back to 7.5; the resulting solutions were then diluted (5) but with some modifications; the rAd5 was comparaand processed as above. Back-titration of samples to pH ble in quality to material purified by cesium chloride 7.5 was carried out because maximum enhancement of density gradient centrifugation (data not shown). The the fluorescence in the presence of DNA occurs at this rAd5 samples were in 20 mM sodium phosphate, 100 pH (3, 4). For the effect-of-temperature experiments, mM NaCl, 2 mM MgCl 2 , 2% sucrose, pH 7.5, and were similar volumes of the same sample were subjected to stored at 080ΠC until use. The stock DNA solution conthe desired temperatures for 10 min using a water bath sisted of calf thymus DNA (Na salt; Sigma Chemical and then were cooled down to room temperature. All Co., St. Louis, MO) in the same buffer as above to a final samples were then diluted and processed as above. concentration of 200 mg/mL. The stock dye solution was
The amount of viral DNA was calculated based on prepared by dissolving H33258 (Sigma Chemical Co.) the following: 1.0 A 260 in the presence of 0.1% SDS Γ
in distilled water to a final concentration of 200 mg/mL 1.1 1 10 12 particles per milliliter (6); adenovirus conand was stored in a dark bottle at 4ΠC for not more tains 13% DNA and 87% protein by weight (7). Abthan 6 months (4). sorbance measurements were carried out using an HP The DNA assay used a Waters (Milford, MA) HPLC 8452A diode array spectrophotometer; all data were system equipped with an autoinjection system and a appropriately blank subtracted and were the average of three determinations. Resource Q anion-exchange