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Enzymatic incorporation and fluorescent labelling of cyclooctyne-modified deoxyuridine triphosphates in DNA
Affiliation:1. Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford OX1 3TA, UK;2. School of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK;3. Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez University, Suez 43721, Egypt;1. Dept. of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK;1. School of Science, Xi’an Jiaotong University, Xi’an 710049, China;2. School of Physics, Xinjiang University, Urumqi 830046, China;1. Sección de Dermatología Pediátrica, Hospital Universitario La Paz, Madrid, España;2. Servicio de Pediatría, Hospital Universitario La Paz, Madrid, España;1. Department of Biotechnology, Chemistry and Pharmacy, University of Siena, I-53100 Siena, Italy;2. Department of Medical Biotechnology, University of Siena, I-53100 Siena, Italy;3. Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, BioLife Science Building, Suite 333, 1900 N 12th Street, Philadelphia, PA 19122, USA;4. Lead Discovery Siena s.r.l, Via Vittorio Alfieri 31, I-53019 Castelnuovo Berardenga, Italy
Abstract:The amino group of 5-aminopropargyl-2′-deoxyuridine-5′-triphosphate was labelled with dibenzocyclooctyne (DIBO) and two derivatives of bicyclo [6.1.0] non-4-yne (BCN) with short and long linkers to produce three different cycloalkyne-modified deoxyuridine triphosphates. BCN was successfully incorporated into DNA at multiple sites by enzyme-mediated primer extension and the polymerase chain reaction (PCR). Efficient fluorescent labelling of the BCN-DNA and DIBO-DNA with Cy3-azide was demonstrated.
Keywords:Copper free click  DNA fluorescent labelling  Cyclooctyne deoxyuridine triphosphates  BCN  DIBO
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