A non-radioactive automated method for DNA sequence determination |
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| Authors: | W Ansorge B S Sproat J Stegemann C Schwager |
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| Affiliation: | 1. University of Amsterdam, Faculty of Medicine, Amsterdam, the Netherlands;2. Department of Ophthalmology, Academic Medical Center, Amsterdam, the Netherlands;3. Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Amsterdam, the Netherlands;4. Retina Total Eye Care, Driebergen, the Netherlands;1. Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan;2. Theoretische Chemie, Physikalisch-Chemisches Institut, Heidelberg University, Im Neuenheimer Feld 229, 69120 Heidelberg, Germany;3. School of Physics and Nuclear Energy Engineering, Beihang University, Beijing 100191, China;4. Cimaina and Dipartimento di Fisica, Università degli Studi di Milano, via Celoria 16, 20133 Milano, Italy;5. European XFEL, Albert-Einstein-Ring 19, Hamburg 22761, Germany;6. Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA;7. Institute for Nuclear Physics, Johann Wolfgang Goethe University Frankfurt, Frankfurt 60438, Germany;8. Department of Physics, Kyoto University, Kyoto 606-8502, Japan;9. UVSOR Facility, Institute for Molecular Science, Okazaki 444-8585, Japan;10. Japan Synchrotron Radiation Research Institute, Sayo, Hyogo 679-5198, Japan;11. National Institute of Advanced Industrial Science and Technology, NMIJ, Tsukuba 305-8568, Japan;1. KACST-Intel Consortium Center of Excellence in Nano-manufacturing Applications (CENA), Riyadh, Saudi Arabia;2. IMEC, Kapeldreef 75, B-3001 Leuven, Belgium;3. Research and Technology Center of Energy, Photovoltaic Department, Borj-Cedria Science and Technology Park, BP 95, 2050, Tunisia;4. University of Carthage, Faculty of Sciences of Bizerta, Tunisia;5. Electrical Engineering Department, College of Engineering & Petroleum, Kuwait University, P.O. Box 5969, 13060 Safat, Kuwait;6. Department of Electrical Engineering (ESAT), K.U. Leuven, 3001 Leuven, Belgium;7. Faculty of Sciences, University of Hasselt, Martelarenlaan 42, 3500 Hasselt, Belgium;1. Instituto de Astronomía y Física del Espacio (IAFE, CONICET-UBA), Buenos Aires, Argentina;2. Instituto de Tecnologías en Detección y Astropartículas (CNEA, CONICET, UNSAM), Centro Atómico Constituyentes, San Martin, Buenos Aires, Argentina |
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| Abstract: | A method and instrument for automated DNA sequencing without radioactivity have been developed. In spite of the success with radioactive labels there are drawbacks attached to the technique, such as hazards in the handling, storage and disposal of radioactive materials, and the considerable cost of the radiolabelled nucleoside triphosphates. In addition, there is deterioration of sample quality with time. A sulphydryl containing M13 sequencing primer has been synthesised and subsequently conjugated with tetramethylrhodamine iodoacetamide. The fluorescent primer is used to generate a nested set of fluorescent DNA fragments. The fluorescent bands are excited by a laser and detected in the gel (detection limit about 0.1 fmol per band) during electrophoresis, and sequence data from the four tracks are transferred directly into a computer. Standard gels, 200 mm wide with 20 sample slots have also been used. The device contains no moving parts. At present 250-300 bases can be read in 6 h. The system is capable of single base resolution at a fragment length of at least 400 bases. |
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