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.
Highlights
Probe-based fluorescence detection is an important tool for imaging and detection of specific DNA and RNA sequences in polymerase chain reaction (PCR) amplicons, fixed cells and tissues [1,2,3]
To improve upon the enzymatic incorporation and fluorescent labelling efficiency that was previously achieved by azidomethyl dUTP (AM dUTP) (Figure 2A), [19] and with ease of chemical synthesis in mind, a new azide-modified triphosphate (AHP dUTP) was designed
Enzymatic incorporation of AHP dUTP was evaluated using a variety of DNA templates and polymerases
Summary
Probe-based fluorescence detection is an important tool for imaging and detection of specific DNA and RNA sequences in polymerase chain reaction (PCR) amplicons, fixed cells and tissues [1,2,3]. Multiple fluorescently-labelled nucleotides have been incorporated directly into DNA probes during PCR or nick-translation using polymerase enzymes [6,7]. Alternative post-labelling strategies have been developed whereby reactive groups are introduced into the DNA probe via a modified triphosphate and serve as substrates for subsequent fluorescent labelling [8,9]. The advantage of this indirect method is that the reactive groups are sterically relatively undemanding, so the modified nucleotides are incorporated efficiently by polymerase enzymes
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