title: Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes.
abstract: A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX.
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford OX1 3TA, UK.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Ren
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford OX1 3TA, UK Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez University, Suez 43721, Egypt.
authors.forename: Afaf H
authors.initials: AH
authors.lastname: El-Sagheer
authors.affiliation: Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford OX1 3TA, UK tom.brown@chem.ox.ac.uk.
authors.forename: Tom
authors.initials: T
authors.lastname: Brown
mesh_terms: D001386:Azides / Q000138:chemical synthesis / Q000737:chemistry*; D061565:Cycloaddition Reaction / Q000379:methods; D004247:DNA / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry*; D003856:Deoxyuracil Nucleotides / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry
chemical_list: D001386:Azides; D015342:DNA Probes; D003856:Deoxyuracil Nucleotides; D005456:Fluorescent Dyes; D009841:Oligonucleotides; C027078:deoxyuridine triphosphate; D004247:DNA; D019809:Streptavidin
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