CHARACTERIZATION OF TO-PRO-3 AS AN INTERCALATOR FOR DOUBLE-STRANDED DNA ANALYSIS WITH RED DIODE LASER-INDUCED FLUORESCENCE DETECTION

We report the analysis of double-stranded DNA (dsDNA) with a blue inte rcalating dye, TO-PRO-3 (TP3) using a visible diode laser-induced fluo rescence (LIF) detection, In the presence of single-stranded DNA (ssDN A), such as pd(A)(40-60) or pd(T)(20-40), TP3 absorption maximum shift s slightly from 631 to 633 nm and, when intercalated to dsDNA, the abs orption maxima shifts to 643 nm. TP3 itself does not fluoresce in the presence of either d(A)(18) or d(T)(18) alone, but the combination of both oligonucleotide species yielded intense fluorescence, about two o rders 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 t he resulting dsDNA-bound TP3 was collected at 670 nm. The capillary el ectrophoretic (CE) separation of fragments from 72 to 1353 basepairs ( bp) of Phi X174/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 are a and fragment size in basepairs were obtained with TP3 and ethidium b romide as the intercalating dyes. TP3-based chemistry along with the d iode laser-induced fluorescence detection system is well-suited for ra pid, high-sensitivity and automated DNA analysis of the PCR-amplified dsDNA products and DNA restriction fragments. (C) 1997 Elsevier Scienc e B.V.