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. 2023 Oct;52(6-7):593-605.
doi: 10.1007/s00249-023-01655-y. Epub 2023 May 4.

Multi intercalators FRET enhanced detection of minute amounts of DNA

Luca Ceresa  1 , Jose Chavez  2 , Magdalena M Bus  3   4 , Bruce Budowle  5   6 , Emma Kitchner  2 , Joseph Kimball  2 , Ignacy Gryczynski  2 , Zygmunt Gryczynski  2
Affiliations

Multi intercalators FRET enhanced detection of minute amounts of DNA

Luca Ceresa et al. Eur Biophys J. 2023 Oct.

Abstract

A novel approach is presented that increases sensitivity and specificity for detecting minimal traces of DNA in liquid and on solid samples. Förster Resonance Energy Transfer (FRET) from YOYO to Ethidium Bromide (EtBr) substantially increases the signal from DNA-bound EtBr highly enhancing sensitivity and specificity for DNA detection. The long fluorescence lifetime of the EtBr acceptor, when bound to DNA, allows for multi-pulse pumping with time gated (MPPTG) detection, which highly increases the detectable signal of DNA-bound EtBr. A straightforward spectra/image subtraction eliminates sample background and allows for a huge increase in the overall detection sensitivity. Using a combination of FRET and MPPTG detection an amount as small as 10 pg of DNA in a microliter sample can be detected without any additional sample purification/manipulation or use of amplification technologies. This amount of DNA is comparable to the DNA content of a one to two human cells. Such a detection method based on simple optics opens the potential for robust, highly sensitive DNA detection/imaging in the field, quick evaluation/sorting (i.e., triaging) of collected DNA samples, and can support various diagnostic assays.

Keywords: DNA detection; DNA intercalator; Förster resonance energy transfer; Multipulsing; Time resolved emission spectra; Time resolved spectroscopy.

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