Toluidine Blue Dye-Binding Method for Measurement of Genomic DNA Extracted From Peripheral Blood Leukocytes.

Abstract

Background: The toluidine blue DNA assay has characteristics that suggest its suitability for concentration determinations of genomic DNA extracted from peripheral blood leukocytes. These include visible wavelength readings, rapid analytical time, useful sensitivity, and low reagent costs. We validated this assay for genomic DNA and devised a simple method to identify extracts that exceed the linear range of the assay. Methods and Results: DNA was prepared from 109 blood samples by a salt extraction method. The toluidine blue assay was linear for DNA concentrations from 0 to 500 ng/µL. By performing bichromatic absorption readings at 570 and 628 nm, extracts with DNA concentrations exceeding the linear range could be identified up to at least 1300 ng/µL. Analytical precision and accuracy studies gave acceptable results. There was excellent agreement between DNA concentrations determined by the toluidine blue assay and ultraviolet spectrophotometry. RNA was confirmed to be significant positive interferent. The detection limit of genomic DNA was determined to be 15 ng/µL. Conclusions: The toluidine blue DNA assay is suitable for measuring DNA concentrations in extracts obtained from peripheral blood. This assay appears to be suitable for automation as a visible spectrophotometric DNA method.