Single molecule DNA sequencing in submicrometer channels: state of the artand future prospects

We demonstrate a new method for single molecule DNA sequencing which is bas ed upon detection and identification of single fluorescently labeled mononu cleotide molecules degraded from DNA-strands in a cone shaped microcapillar y with an inner diameter of 0.5 mum. The DNA was attached at an optical fib er via streptavidin/biotin binding and placed similar to 50 mum in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-m er model DNA sequence used in the experiments contained 6 fluorescently lab eled cytosine and uridine residues, respectively, at well defined positions . The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic fo rces. Adsorption of mononucleotide molecules onto the capillary walls as we ll as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvi nyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excita tion of the labeled mononucleotide molecules a short-pulse diode laser emit ting at 638 nm with a repetition rate of 57 MHz was applied. We report on e xperiments where single-stranded model DNA molecules each containing 6 fluo rescently labeled dCTP and dUTP residues were attached at the tip of a fibe r, transferred into the microcapillary and degraded by addition of exonucle ase I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP an d 43 MR121-dUMP) during 400 s and identified due to the characteristic fluo rescence decay time of the labels of 1.43 +/-0.19 ns (Cy5-dCMP), and 2.35 /-0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-strand ed labeled DNA molecules was determined to 3-24 Hz under the applied experi mental conditions. In addition, the observed burst count rate (signals/s) i ndicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA. (C) 2001 Elsevier Science B.V. All rights reserved.