Characterization of TO-PRO-3 as an intercalator for double-stranded DNA analysis with red diode laser-induced fluorescence detection

Abstract

We report the analysis of double-stranded DNA (dsDNA) with a blue intercalating dye, TO-PRO-3 (TP3) using a visible diode laser-induced fluorescence (LIF) detection. In the presence of single-stranded DNA (ssDNA), such as pd(A) 40–60 or pd(T) 20–40, TP3 adsorption maximum shifts slightly from 631 to 633 nm and, when intercalated to dsDNA, the absorption maxima shifts to 643 nm. TP3 itself does not fluoresce in the presence of eithet d(A)_ 18 or d(T) 18 alone, but the combination of both oliginucleotide species yielded intense fluorescence, about two orders of magnitude higher. The LIF detection was based on the use of a 2.5-mW diode laser emitting light at 635 nm and the fluorescence of the resulting dsDNA-bound TP3 was collected at 670 nm. The capillary electrophoretic (CE) separation of fragments from 72 to 1353 basepairs (bp) of ΦX174/ HaeIII digest were well-resolved within 10 min in the gel-buffer system with TP3. The application of TP3 as an intercalating dye for the analysis of dsDNA fragments produced by polymerase chain reaction (PCR) was examined. Excellent correlations between CE-LIF area and fragment size in basepairs were obtained with TP3 and ethidium bromide as the intercalating dyes. TP3-based chemistry along with the diode laser indiced fluorescence detection system is well-suited for rapid, high-sensitivity and automated DNA analysis of the PCR-amplified dsDNA products and DNA restriction fragments.