Real‐time Detection of Nucleotide Incorporation During Complementary DNA Strand Synthesis

Krieg, Alexander; Laib, Stephan; Ruckstuhl, Thomas; Seeger, Stefan

doi:10.1002/cbic.200200549

Cited by 14 publications

(13 citation statements)

References 24 publications

(14 reference statements)

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“…Recent examples for other evanescent field fluorescent techniques that were used to monitor DNA polymerase and telomerase action are super critical angle fluorescence (SAF) detection (22,23), optical fibers (24), zero-mode waveguides (25) and a mini-DNA sequencing concept based on the single molecule fluorescence microscopy (26). In the present work, we have derived binding and catalytic constants for the KF by using SPFS, with particular focus on the merit of the primer extension reaction for the sensitive detection of single base point mutation and the added value obtained by probing interfacial reflectivity and fluorescence changes simultaneously.…”

Section: Introductionmentioning

confidence: 99%

Surface plasmon field-enhanced fluorescence spectroscopy studies of primer extension reactions

Stengel

Knoll

2005

Nucleic Acids Research

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Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) utilizes the evanescent electromagnetic field of a surface plasmon to excite chromophors in close proximity to the surface. While conventional surface plasmon resonance spectroscopy allows the observation of surface reactions by means of refractive index changes, SPFS additionally provides a channel for the read-out of fluorescence changes. Thus, the detection limit for low mass compounds, whose adsorption is only accompanied by small refractive index changes, can be substantially improved by fluorescent labeling. In this study, we present the first example that utilizes SPFS to follow the dynamics of an enzymatic reaction. The elongation of surface-tethered DNA has been observed by the incorporation of Cy5-labeled nucleotides into the nascent strand by the action of DNA polymerase I (Klenow fragment). The technique offers a rapid way to determine the binding constant and the catalytic activity of a DNA processing enzyme, here exemplified by the Klenow fragment. Furthermore, the effect of mispaired bases in the primer/template duplex and the influence of different label densities have been studied. The resulting sensitivity for nucleotide incorporation, being in the femtomolar regime, combined with the specificity of the enzyme for fully complementary DNA duplexes suggest the application of this assay as a powerful tool for DNA detection.

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Section: Introductionmentioning

confidence: 99%

Surface plasmon field-enhanced fluorescence spectroscopy studies of primer extension reactions

Stengel

Knoll

2005

Nucleic Acids Research

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“…In this case, primer, dNTPs and polymerase were applied to a template‐coated coverslip simultaneously. This approach is reasonable since hybridisation reactions under comparable conditions were found to be completed within approximately 5 min 41. A possible drawback of method 2 is that the intensity increase observed after addition of Cy5‐dCTPs could superpose on the increase caused by primer elongation.…”

Section: Resultsmentioning

confidence: 95%

“…The remaining sequence of the template was chosen to have no correspondence to the primer sequence and to contain five guanine residues. In the case of successful corresponding strand synthesis, five Cy5‐dCTPs are expected to be incorporated into the dsDNA at the interface 41…”

Section: Resultsmentioning

confidence: 99%

Fast Detection of Single Nucleotide Polymorphisms (SNPs) by Primer Elongation with Monitoring of Supercritical‐Angle Fluorescence

et al. 2004

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We describe the rapid detection of single nucleotide polymorphisms (SNPs) by real-time observation of primer elongation. The enzymatic elongation of surface-bound primers is monitored by detecting the increase of surface-bound fluorescence caused by the incorporation of Cy5-labelled deoxycytidine 5'-triphosphate residues (Cy5-dCTPs) into the corresponding strand. In order to discriminate against the fluorescence from unbound Cy5-dCTPs, the detection volume was restricted to the surface by collecting supercritical-angle fluorescence. The efficiency of enzymatic double-stranded DNA synthesis is governed by the complementarity of the primer and template. An SNP in the sequence of the primer obstructs its elongation increasingly with decreased distance of the mismatch to the 3' end of the primer. By real-time fluorescence detection during primer elongation, SNPs can be detected within a few minutes, which is significantly faster than in experiments where the fluorescence is measured after completion of the reaction. We demonstrate the efficiency of the method by detecting an SNP in the ErbB2 gene that is involved in causing a higher risk of breast cancer.

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“…Cy5-dCTP and Cy5-dUTP (ribonucleic acid pendant of dTTP). The incorporation of Cy5 into the surface bound complexes was detected by measuring the supercritical angle fluorescence (SAF) emission in real time with a biosensor of custom design [10].…”

Section: Introductionmentioning

confidence: 99%

Real-Time Detection of Polymerase Activity Using Supercritical Angle Fluorescence

Krieg

Ruckstuhl

Laib

et al. 2004

Journal of Fluorescence

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We investigated the incorporation efficiencies of different fluorescently labelled dNTPs with polymerases by complementary strand synthesis. For this reason single stranded DNA was immobilized on a coverslip and the increase of fluorescence due to the synthesis of the corresponding strand with tagged dNTPs was detected with a supercritical angle fluorescence biosensor in real-time. By comparison of the observed signal intensities it was possible to conclude that the system Cy5-dCTP-Klenow (exonuclease free) fragment gives the best incorporation yield of the investigated enzymes and dNTPs.

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Real‐time Detection of Nucleotide Incorporation During Complementary DNA Strand Synthesis

Cited by 14 publications

References 24 publications

Surface plasmon field-enhanced fluorescence spectroscopy studies of primer extension reactions

Surface plasmon field-enhanced fluorescence spectroscopy studies of primer extension reactions

Fast Detection of Single Nucleotide Polymorphisms (SNPs) by Primer Elongation with Monitoring of Supercritical‐Angle Fluorescence

Real-Time Detection of Polymerase Activity Using Supercritical Angle Fluorescence

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