CHARACTERIZATION OF DNA SIZE DETERMINATION OF SMALL FRAGMENTS BY FLOW-CYTOMETRY

DNA fragment lengths were determined using the intensity of fluorescen t bursts from single fragments stained with a thiazole orange derivati ve, The individual stained fragments were introduced into a sheath now cuvette and passed through a low-power (30 mW), continuous-wave laser beam with transit times in the range 3-5 ms. As little as 50 fg of DN A was analyzed at a rate of 40 fragments/s for times ranging from 1 to 15 min. a detectable lower size limit of 1.5 kilobase pairs (kbp) was demonstrated, and a linear relationship between fluorescence intensit y and fragment length was observed. Issues relating to size resolution in the 2-50 kbp range are discussed.