Detection of individual oligonucleotide pairing by single-molecule microscopy

Hybridization of 20mer probe oligonucleotides to complementary, surface-imm obilized target oligonucleotides was visualized on a single-molecule basis by fluorescence microscopy. Coincident determination of the positions of bo th the target and the probe oligonucleotides using dual-wavelength fluoresc ence labeling allowed for highly reliable discrimination of specifically bo und probe molecules from those being physisorbed. The figures of merit of t he 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 D NA fragments per second. The probability for false negative events is limit ed by the biochemical binding probability of short oligonucleotides. The po tentials and limitations of this methodology for single-cell single-DNA ana lysis are discussed.