Abstract
We have developed an automated DNA sequenator using a real-time fluorescence detection method, and achieved excellent sensitivity and rapid analysis by optimizing operating conditions. The sequenator uses unicolor labeling and four-track electrophoresis. Fluoresceine isothiocyanate(FITC)-labeled primers are used to produce labeled DNA families. The fluorescent bands are excited by an Ar laser (488 nm, 10 mW) introduced from the side of a thin gel during electrophoresis. Analysis, with an accuracy greater than 99%, consumes 0.01–0.04 picomole sample for each DNA family, which is almost comparable to the amount used in conventional autoradiography. With the optimized electrophoresis conditions reported here, four hundred bases of DNA can be analyzed within two and a half hours using a four percent polyacrylamide gel.
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Kambara, H., Nishikawa, T., Katayama, Y. et al. Optimization of Parameters in a DNA Sequenator Using Fluorescence Detection. Nat Biotechnol 6, 816–821 (1988). https://doi.org/10.1038/nbt0788-816
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DOI: https://doi.org/10.1038/nbt0788-816
