A VISIBLE SPECTROPHOTOMETRIC ASSAY FOR SUBMICROGRAM QUANTITIES OF DNA, INCLUDING PCR-AMPLIFIED DNA

The use of the cationic thiazine dye toluidine blue for solution measu rements of DNA in the submicrogram range was studied with particular r egard to the measurement of the small quantities of DNA synthesized in a typical polymerase chain reaction (PCR). Tolouidine blue shows a de crease in absorption at its wavelength of maximal absorption, 628 nm, on binding DNA in low concentrations. Using toluidine blue at 2 x 10(- 5) M, there was a linear response to increasing DNA concentrations up to 2000 ng of DNA in a 500 mu l assay. The lower limit of detection wa s 24 ng of DNA. The assay was found to be sufficiently sensitive to me asure the DNA produced in a PCR, and to distinguish PCRs that amplifie d a product from those in which amplification did not occur. The effec ts of selected metal ions and nonmetals on the assay were assessed. Of the compounds tested only sodium dodecyl sulfate was found to be inco mpatible with the assay. Compared to other colorimetric DNA assays, th e 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 measure ment of the microgram quantities of DNA produced in a typical PCR. (C) 1995 Academic Press, Inc.