Amyloglucosidase suppresses interference by glycogen in the quantification of DNA using the Hoechst 33258 dye.

The Hoechst dye fluorescence technique, which is used for the quantitative determination of DNA (12), requires high saline concentrations at neutral pH to reach maximal fluorescence of the DNA-dye complex and to render the DNA fully accessible to the dye by dissociating foreign molecules (3,12). In conventional assays, the DNA is alcohol-precipitated and then resuspended in buffer containing 2 M NaCl (12). The procedure has been modified for low DNA concentrations (<1 μg/mL) by adding a co-precipitating macromolecule such as glycogen, linear polyacrylamide, tRNA, or spermidine before the alcohol step to improve precipitation and recovery of DNA (8,10). A combination of the co-precipitation and fluorescence techniques is convenient for quantitative DNA determinations in samples containing low DNA concentrations. However, the Hoechst 33258 dye can bind to compounds other than nucleic acids (2,4) and hence can cause nonspecific fluorescence. When proteins or polysaccharides such as glycogen are mixed with the dye at a weight ratio equivalent to that of DNA in cellular homogenates (4), the nonspecific fluorescence represents less than 1% of the fluorescence attributable to the DNA-dye complex. A substantially higher error is to be expected when proteins or polysaccharides are present at concentrations higher than that of DNA, for example, when a carrier is used for DNA precipitation. Between 10 and 30 μg carrier are required per milliliter to precipitate picogram or nanogram quantities of DNA (8,10,14). Here we investigated the effect of carrier on the DNA-Hoechst 33258 dye fluorescence, which was measured when DNA was precipitated in the presence of glycogen. We then developed a method to degrade the glycogen enzymatically (13) after the precipitation of DNA and before the fluorescence measurement. We used the Hoechst 33258 dye (2′[4-hydroxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazole), lowgrade glycogen type VII (from mussel, approximately 108 Da) (5) and genomic E. coli DNA type VIII (all purchased from Sigma-Aldrich, Taufkirchen, Germany). Glycogen for molecular biology (from mussel) and amyloglucosidase (approximately 105 Da) were obtained from Roche Applied Science (Mannheim, Germany). All other chemicals were of analytical grade. The dye was dissolved at 0.2 μg/μL in water, passed through a 0.2-μm filter, aliquoted, and stored in the dark at -20°C. Amyloglucosidase (amylo-α1,4-α-1,6-glucosidase) was prepared at 1 μg/μL in 0.1 M sodium acetate, 2.5 mM Tris, pH 4.8 (13), and stored at -20°C. The DNA was dissolved in filtered TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and stored at 4°C. The DNA stock solution contained fragments of less than 25 kb, their bulk ranging in size from 10 to 25 kb. Fragment sizes were determined by ultracentrifugation in 5%–25% sucrose density gradients or by electrophoresis on 0.7% agarose gels. The DNA of bacteriophage λ cleaved with Eco130I (StyI) and MluI was co-electrophoresed as size marker. Fluorometric assays were carried out on resuspended DNA (Table 1), diluted in 700 μL PBS (50 mM NaH2P O4, 2 M NaCl, pH 7.4), and then mixed with 7.5 μL freshly prepared Hoechst 33258 dye solution at 0.02 μg/μL in water. Fluorescence was measured in a model LS 50B luminescence spectrophotometer (Perkin Elmer Ltd., Buckinghamshire, UK), adjusting excitation at 350 nm and emission at 460 nm. The excitation slit width was set to 2.5 nm and the emission slit to 7.0 nm. Mean values and simple regressions were calculated with a confidence interval of 95% for three replicate measurements. In addition, fluorescence was measured in the presence of increasing concentrations of glycogen type VII, which was thought to contain traces of contaminating DNA (10), and of glycogen for molecular biology, which was free of nucleic acids. As Benchmarks