Towards a general procedure for sequencing single DNA molecules

In this paper we report on the latest technical advances towards single mol ecule sequencing, a useful method currently developed especially for fast a nd easy de novo sequencing. Different approaches for complete labeling of D NA with fluorescent dyes are described. In addition, the experimental set-u p for the sequencing process is shown. We demonstrate the ability to purify the buffer and enzyme solutions. Inorganic buffers were purified down to a t least 20 fM of remaining fluorescent impurities. The exonuclease buffer s olution could be cleaned down to 0.8 pM whereby its full activity was kept. Finally, we show a selection procedure for beads and present the data of a model experiment, in which immobilized DNA is degraded by an exonuclease w ithin a polymethylmethacrylate (PMMA) microstructure. Furthermore, the math ematical processing of the obtained raw data is described. A first complete experimental cycle is shown, combining all preparatory steps which are nec essary for single molecule sequencing in microstructures. (C) 2001 Elsevier Science B.V. All rights reserved.