Towards a general procedure for sequencing single DNA molecules

• Published in: Journal of biotechnology 2001-04, Vol.86 (3), p.255-267
• Main Authors: Stephan, Jens, Dörre, Klaus, Brakmann, Susanne, Winkler, Thorsten, Wetzel, Timm, Lapczyna, Markus, Stuke, Michael, Angerer, Bernhard, Ankenbauer, Waltraut, Földes-Papp, Zeno, Rigler, Rudolf, Eigen, Manfred
• Format: Article
• Language: English
• Subjects: Biochemistry - methods; Buffers; de novo DNA sequencing; DNA - analysis; DNA - chemistry; DNA labeling; DNA sequencing; DNA-Directed DNA Polymerase - chemistry; Exonucleolytic degradation; Fluorescent Dyes - chemistry; Medicin och hälsovetenskap; Microspheres; Microstructures; Polymethyl Methacrylate; Sequence Analysis, DNA; Single molecule detection
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/S0168-1656(00)00417-X

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.