3-1.3.5 Fluorometric Quantification:

Fluorometric method applies fluorescence dyes to detect the presence and concentration of a class of nucleic acid (DNA or RNA). This method is more sensitive and less prone to contaminants than UV spectroscopy.

An assay using Hoechst 33258 dye is specific for DNA because it is less sensitive to detect RNA. This assay is commonly used for rapid measurement of low quantities of DNA, with a detection limit of ~ 1 ng DNA. It is useful for the measurement of both small and large amounts of DNA (verifying DNA concentrations prior to performing electrophoretic separations and Southern blots) because this assay accurately quantifies a broad range of DNA concentrations from10 ng/ml to15 μg/ml. The Hoechst 33258 assay can also be employed for measuring products of the polymerase chain reaction (PCR) synthesis.

Hoechst 33258 is non-intercalating reagent and binds to the minor groove of the DNA with a preference for AT sequences (Portugal and Waring, 1988). The binding to the minor groove has is dependent upon a combination of structural preferences (eg., the minor groove with a series of contiguous AT base pairs is more narrow). (Neidle (2001) ,Like other minor groove binding ligands, Hoechst 33258 is positively charged and thus form electrostatic interaction with the negative potential of stretch of AT base pairs. Upon binding to the minor groove of the double helix DNA, the fluorescence characteristics of Hoechst 33258 change dramatically, showing a large increase in emission at ~ 458 nm.

According to Daxhelet et al, the fluorochrome 4' ,6-diamidino-2-phenylindole (DAPI) has similar characteristics to H33258 and binds to the minor groove as well. DAPI is also appropriate for DNA or RNA quantitation, although it is not as commonly used as Hoechst 33258. DAPI is excited with a peak at 344 nm. Emission is detected at around 466 nm for DNA, similar to Hoechst 33258 but for RNA the peak shifts to ~500 nm.