Many techniques of cellular and molecular biology require the ability to quantitate dsDNA in large numbers of samples at sensitivities that only require a small amount of the total sample. Although there are many different methods to quantitate DNA, most methods have disadvantages that preclude their use in some applications. Absorbance measurements at 260 nm (A260) is the most commonly used method for DNA concentration determination, but it suffers from the interfering absorbance of contaminating molecules (1). Many of these contaminants, which include nucleotides, RNA, EDTA and phenol, are commonly found in nucleic acid preparations. The fluorescent bisbenzimide (Hoechst) dyes circumvent many of these problems. Hoechst 33258 dye is relatively selective for dsDNA and in high salt does not show fluorescent enhancement in the presence of either protein or RNA. The dye, weakly fluorescent itself in solution, binds specifically to the A-T base pairs in dsDNA resulting in an increase in fluorescence and a shift in the emission maximum from 500 to 460 nm (2, 3). The use of Hoechst 33258 in conjunction with the BioTek Microplate Fluorescence Readers offer high specificity, as well as high sensitivity for dsDNA quantitation.
For most investigators using the dye concentrations described by Labarca and Paigen (3) are adequate, but optimal concentrations can be determined empirically. Using a Hoechst 33258 dye concentration of 1.0 mg/ml would be expected give a linear response to a wider range of DNA concentrations than lower dye concentrations. Alternatively for low levels of DNA the use of 0.1 mg/ml dye concentrations may be more appropriate, as background fluorescence is reduced. The caveat of this condition being a decrease in fluorescent signal response is resulting in a flatter dose response and saturation at lower dsDNA.
In terms of reagents this assay only requires two stock solutions. The assay buffer is composed of 2M NaCl and 50mM NaH2PO4, pH 7.4 and sterilized by autoclaving, while the stock dye solution is (1 mg/ml Hoechst dye in distilled H2O) and sterilized by filtration through a 0.22 mm filter. Both are stored at 4°C. Working solution is prepared by mixing 1 µL of dye per 1 mL of assay buffer. The fluorescence is measured in a microplate reader with a 360/40 excitation filter and a 460/40 emission filter.
This assay offers good sensitivity at a low price. As a fluorescent assay it is certainly much more sensitive than a direct absorbance at 260 nm determination, but it does require that a standard curve be used. While it is not as sensitive as a PicoGreen® assay, because the reagents, particularly the dye, are not proprietary they can be purchased at a very reasonable cost to the investigator, resulting in a cost per assay being less than $0.01. Other than the dye the assay only requires sodium phosphate for buffering and sodium chloride and the stock solutions last for extended periods of time if stored at 5°C. For the cost conscience lab that is not afraid to make their assay reagents, this is a wonderful assay and I highly recommend it. For more information regarding this DNA quantitation application using microplates check out the Hoechst application note on the BioTek website or the references listed below..
(1) Maniatis, T., E.F. Fritsch, and J. Sambrook (1982) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Springs Harbor, NY.
(2) Daxhelet, G.A., M.M. Coene, P.P. Hoet, and C.G. Cocito (1989) Analytical Biochemistry 179:401-403.
(3) Labarca, C. and K. Paigen (1980). Analytical Biochemistry 102:344-352.
By, BioTek Instruments