A Visible Spectrophotometric Assay for Submicrogram Quantities of DNA, Including PCR-Amplified DNA

The use of the cationic thiazine dye toluidine blue for solution measurements of DNA in the submicrogram range was studied with particular regard to the measurement of the small quantities of DNA synthesized in a typical polymerase chain reaction (PCR). Tolouidine blue shows a decrease in absorption at its wavelength of maximal absorption, 628 nm, on binding DNA in low concentrations. Using toluidine blue at 2 × 10^-5 M, there was a linear response to increasing DNA concentrations up to 2000 ng of DNA in a 500 μl assay. The lower limit of detection was 24 ng of DNA. The assay was found to be sufficiently sensitive to measure the DNA produced in a PCR, and to distinguish PCRs that amplified a product from those in which amplification did not occur. The effects of selected metal ions and nonmetals on the assay were assessed. Of the compounds tested only sodium dodecyl sulfate was found to be incompatible with the assay. Compared to other colorimetric DNA assays, the toluidine blue assay is 10- to 100-fold more sensitive, approaching the sensitivity achieved by fluorometric DNA assays. The color change on binding DNA is immediate, and takes place at room temperature, thus allowing for rapid measurements to be made. The toluidine blue assay extends the useful range of visible spectrophotometric DNA assays well into the submicrogram range and is suitable for detection and measurement of the microgram quantities of DNA produced in a typical PCR.