Detection of individual oligonucleotide pairing by single-molecule microscopy

Hybridization of 20mer probe oligonucleotides to complementary, surface-immobilized target oligonucleotides was visualized on a single-molecule basis by fluorescence microscopy. Coincident determination of the positions of both the target and the probe oligonucleotides using dual-wavelength fluorescence labeling allowed for highly reliable discrimination of specifically bound probe molecules from those being physisorbed. The figures of merit of the assay are characterized by the low probability for false positive (10(-4)) events and the high speed for detection of up to hundreds of different DNA fragments per second. The probability for false negative events is limited by the biochemical binding probability of short oligonucleotides. The potentials and limitations of this methodology for single-cell single-DNA analysis are discussed.