High-sensitivity two-color detection of double-stranded DNA with a confocal fluorescence gel scanner using ethidium homodimer and thiazole orange

Abstract

Ethidium homodimer (EthD; lambda Fmax 620 nm) at EthD:DNA ratios up to 1 dye:4-5 bp forms stable fluorescent complexes with double-stranded DNA (dsDNA) which can be detected with high sensitivity using a confocal fluorescence gel scanner (Glazer, A.N., Peck, K. & Mathies, R.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3851-3855). However, on incubation with unlabeled DNA partial migration of EthD takes place from its complex with dsDNA to the unlabeled DNA. It is shown here that this migration is dependent on the fractional occupancy of intercalating sites in the original dsDNA-EthD complex and that there is no detectable transfer from dsDNA-EthD complexes formed at 50 bp: 1 dye. The monointercalator thiazole orange (TO; lambda Fmax 530 nm) forms readily dissociable complexes with dsDNA with a large fluorescence enhancement on binding (Lee, L.G., Chen, C. & Liu, L.A. (1986) Cytometry 7, 508-517). However, a large molar excess of TO does not displace EthD from its complex with dsDNA. When TO and EthD are bound to the same dsDNA molecule, excitation of TO leads to efficient energy transfer from TO to EthD. This observation shows the practicability of 'sensitizing' EthD fluorescence with a second intercalating dye having a very high absorption coefficient and efficient energy transfer characteristics. Electrophoresis on agarose gels, with TO in the buffer, of preformed linearized M13mp18 DNA-EthD complex together with unlabeled linearized pBR322 permits sensitive fluorescence detection in the same lane of pBR322 DNA-TO complex at 530 nm and of M13mp18 DNA-EthD complex at 620 nm. These observations lay the groundwork for the use of stable DNA-dye intercalation complexes carrying hundreds of chromophores in two-color applications such as the physical mapping of chromosomes.