Ultrasensitive detection of single DNA molecules

Abstract

We report on the detection of single lambda DNA molecules in flowing TBE buffer. DNA molecules at 10−14 M bound the fluorescent dye ethidium bromide at 10−10 M and were detected by using our recently developed technique for single molecule detection. This technique involves repetitive excitation of individual fluorophores with a frequency-doubled Nd:YAG laser, fluorescence detection with a microchannel-plate photo-multiplier based time-correlated single-photon counter, and signal processing with time-gated discrimination electronics. This detection takes 50 ms in a sample volume of 3.5 pL through which the DNA molecules pass at a rate of about 1 per second. The fluorescence lifetime of ethidium bromide increases from 1 to 24 ns when it becomes intercalated in DNA. To ensure that only DNA molecules were detected, we set a time window from 5 to 10 ns after the arrival of the laser pulse, thus rejecting both prompt scattering and fluorescence from free EtdBr molecules. The present method is at least 1000 times as sensitive as current flow cytometry methods. Quantitation of the fluorescence intensity will allow us to determine the lengths of individual DNA molecules, which can lead to the development of separation and size sorting techniques that are much faster than present methods.