Thorsten Winkler

Towards a general procedure for sequencing single DNA molecules.

In this paper we report on the latest technical advances towards single molecule sequencing, a useful method currently developed especially for fast and easy de novo sequencing. Different approaches for complete labeling of DNA with fluorescent dyes are described. In addition, the experimental set-up for the sequencing process is shown. We demonstrate the ability to purify the buffer and enzyme solutions. Inorganic buffers were purified down to at least 20 fM of remaining fluorescent impurities. The exonuclease buffer solution 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 within a polymethylmethacrylate (PMMA) microstructure. Furthermore, the mathematical processing of the obtained raw data is described. A first complete experimental cycle is shown, combining all preparatory steps which are necessary for single molecule sequencing in microstructures.

Dual-Color Confocal Fluorescence Spectroscopy and its Application in Biotechnology

In recent years, fluorescence correlation spectroscopy (FCS) has become an attractive analytical tool for the investigation of biomolecular processes at the single molecular level. This method was invented in the early 1970s by groups at Cornell University, Ithaca, N.Y. [9.1,9.2], and at the Karolinska Institute, Stockholm [9.3,9.4]. During the 1990s, modern confocal optics, new dyes as efficient fluorescent probes, sensitive photon detectors, and fast data processing tools have been introduced, mainly by Rigler and Eigen [9.5,9.6]. Due to these improvements, FCS permits the observation of the dynamics of single molecules in real time while they pass an open volume element of less than a femtoliter, i.e. the size of a common bacterial cell. Nowadays, FCS has found its way into several laboratories and companies all over the world as a tool for basic research as well as for industrial applications such as drug screening.