FLUORESCENCE DETECTION AND SIZE MEASUREMENT OF SINGLE DNA-MOLECULES

We have developed a technique for the detection and size discrimination of single DNA molecules in a hydrodynamically focused flowing solution. Double-stranded lambda DNA molecules at 3 X 10(-15) M were stained with the fluorescent dye TOTO-1 and were individually detected. The technique makes use of a frequency-doubled mode-locked Nd:YAG laser to repetitively excite the molecules as they traverse the tightly focused laser beam. The flowing sample solution was hydrodynamically focused down to a 20-mum-diameter stream by a rapidly flowing water sheath. The sheath flow technique is well suited for laser-induced fluorescence detection of small-volume, low-concentration samples. The emitted fluorescence photon burst originating from a single DNA molecule was detected with a microchannel plate photomultiplier based single-photon counter, which used time-gated electronics for Raman and Rayleigh scattering rejection. In addition, a mixture of lambda DNA and smaller single-cut fragments has been simultaneously detected and identified by size. The advantages over other techniques for the detection and size determination of DNA fragments are discussed.