Fluorescence Lifetime for On-the-Fly Multiplex Detection of DNA Restriction Fragments in Capillary Electrophoresis

Abstract

This paper describes the first use of frequency-domain fluorescence lifetime for multiplex detection of DNA restriction fragments in capillary electrophoresis (CE). The fragments were labeled with monomeric intercalating dyes that can be excited by either the 488- or 514-nm line of an argon ion laser and have lifetimes in the range of 0.5-2.5 ns. We were able to achieve multiplex lifetime detection in the CE separation of a restriction fragment digest and a DNA size ladder in the same run, for fragments shorter than 700 bp. Different gel buffer systems, including a modified polyacrylamide gel and several tris-borate-EDTA/hydroxyethylcellulose (TBE/HEC) gels, were investigated for separation and detection of the dye-labeled DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a gel containing 1% high molecular weight (90,000-105,000) HEC and 0.3% low molecular weight (24,000-27,000) HEC in TBE buffer.

Mono‐ and bis‐intercalating dyes for multiplex fluorescence lifetime detection of DNA restriction fragments in capillary electrophoresis

“…These fragments migrated as a large peak near 700 s for Dye 50 and 800 s for the bis-intercalators. A similar lack of resolution of short fragments was previously observed for the same digest that was labeled with mono-intercalating dyes [8] and is attributed to limitations of the gel matrix. In both the present and previous studies, an anomalously long lifetime is recovered across the broad peak of unresolved fragments.…”

“…Multiplex fluorescence lifetime detection of CE-separated DNA fragments was previously described using monointercalating dyes containing one or two positive charges [8]. The purpose of the present work was to determine if it would be advantageous to use more highly charged (31 or 41) mono-and bis-intercalating dyes.…”

“…In the previous study of monomeric intercalating dyes [8], it was found that dyes can be exchanged among fragments. If one of the dyes has a stronger DNA binding affinity than the other, it may replace the other dye in the fragments.…”

“…It should be noted that the migration times for the mono-intercalating, Dye 50-labeled fragments were shorter than those for the bis-intercalators; the latter were similar to each other. It has been demonstrated for mono-intercalating, monomeric dyes that dye:DNA base pair (bp) ratio affects both CE resolution of fragment peaks and lifetime detection [8]. At a ratio of 1 dye per 20 bp (Fig.…”

“…An alternative approach to multiplex detection in CE is to use fluorescence lifetime detection, which has been performed using both time domain [6] and frequency domain [7] techniques. Fluorescence lifetime detection has been applied to multiplex detection of DNA restriction fragments [8] and DNA sequencing [9][10][11]. The advantages of frequency domain over time domain for lifetime detection, as well as the advantages of lifetime over fluorescence color detection, have been discussed elsewhere [7,12].…”