Count-based quantitation of trace level macro-DNA molecules

Abstract

Quantitation of the trace amount of DNA by counting individual DNA molecules using a high-sensitivity flow cytometric setup has been developed and evaluated for the purpose of establishing a reference analytical procedure. Model DNA molecules, represented by lambda (λ) viral DNA (48 502 bp, double-stranded), were electro-focused to form a tightly bound flow stream on a detection point situated on the centre axis of fused silica tubing measuring 50 µm × 50 µm. The individual DNA particles that were stained with a fluorescent dye were detected individually with a high-sensitivity laser-induced fluorescence (LIF) detection system. Assuming all DNA particles in a given sample volume were detected and counted ('exhaustive counting'), its molar concentration can be calculated without the need for calibration materials. The validity of the proposed measurement method was thoroughly examined and discussed.