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.
Stephan
J
J
Max-Planck-Institut für biophysikalische Chemie, Abteilung Biochemische Kinetik, Am Fassberg 2, D-37077 Göttingen, Germany. jstepha@gwdg.de
Dörre
K
K
Brakmann
S
S
Winkler
T
T
Wetzel
T
T
Lapczyna
M
M
Stuke
M
M
Angerer
B
B
Ankenbauer
W
W
Földes-Papp
Z
Z
Rigler
R
R
Eigen
M
M
D001671:Biochemistry / Q000379:methods*; D002021:Buffers; D004247:DNA / Q000032:analysis / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D008863:Microspheres; D019904:Polymethyl Methacrylate; D017422:Sequence Analysis, DNA
D002021:Buffers; D005456:Fluorescent Dyes; D004247:DNA; D019904:Polymethyl Methacrylate; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase