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.

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The degree and characteristics of physical degradation of macro-DNA molecules by common laboratory manipulations are reported. With linearized lambda-phage viral DNA as the model DNA, fragmentation of macro-DNA by various indispensable laboratory manipulations were investigated using a high sensitivity flow cytometric setup. Investigated manipulations included pipetting, vortexing, rocking, freezethawing, ultrasonication and ultrafiltration. "Exhaustive counting" of the intact lambda DNA molecules following such manipulations enabled a quantitative assessment of the resulting fragmentation, which also revealed the type of degradation reflected in the fragmentation patterns. The use of high sensitivity flow cytometry was especially suited to investigate the degradation of dilute DNA solutions that may not be suitable for analysis using traditional methods. Notable findings of this study included: the boarderline-size of DNA chains in terms of susceptibility to shear stresses by such manipulations; discernable instability of nicked DNAs; shattering-fragmentation of DNAs by freezethawing or ultrasonication; effectiveness of some protection media; marked "self-protection effect" of concentrated DNA solutions. These findings support and refine our traditional knowledge on how to maintain the physical integrity of macro-DNA molecules against inevitable laboratory manipulations.

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