2003
DOI: 10.1021/ja034716g
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Approaching Real-Time Molecular Diagnostics:  Single-Pair Fluorescence Resonance Energy Transfer (spFRET) Detection for the Analysis of Low Abundant Point Mutations in K-ras Oncogenes

Abstract: The aim of this study was to develop new strategies for analyzing molecular signatures of disease states approaching real-time using single pair fluorescence resonance energy transfer (spFRET) to rapidly detect point mutations in unamplified genomic DNA. In addition, the detection process was required to discriminate between normal and mutant (minority) DNAs in heterogeneous populations. The discrimination was carried out using allele-specific primers, which flanked the point mutation in the target gene and we… Show more

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Cited by 146 publications
(102 citation statements)
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“…The signal from the DNA target is boosted without the use of a polymerase, however the target is still being enzymatically amplified. This technique allows for detailed analysis of single-nucleotide polymorphisms (SNPs) [7].…”
Section: Enzyme Labeling Strategies Without Polymerase Target Amplifimentioning
confidence: 99%
“…The signal from the DNA target is boosted without the use of a polymerase, however the target is still being enzymatically amplified. This technique allows for detailed analysis of single-nucleotide polymorphisms (SNPs) [7].…”
Section: Enzyme Labeling Strategies Without Polymerase Target Amplifimentioning
confidence: 99%
“…Furthermore, as only one excitation wavelength is used, there is no loss of detection efficiency in confocal measurements due to imperfect overlap of laser sources, although the overlap of detection volumes defined by separate pinholes must still be optimised. However, direct excitation of the acceptor at donor's excitation wavelength and spectral crosstalk from donor emission can lead to weak signals in the An assay based on spFRET detection of a 'reverse Molecular Beacon' formed by target-dependent ligation of a pair of labelled oligonucleotides has been described by Wabuyele et al 133 The two probes each contain a target-binding sequence and a 10 nt arm sequence complementary to each other. These arm sequences do not hybridise to form an intermolecular duplex (due to its low thermodynamic stability), but following ligation and re-equilibration a conformational re-organisation into an intramolecular hairpinloop occurs, placing the donor (Cy5) in close proximity to the acceptor (Cy5.5), leading to FRET (Fig.…”
Section: Fret-based Detectionmentioning
confidence: 99%
“…Applications where the viability of near-IR fluorescence has been demonstrated include DNA sequencing (4)(5)(6), detecting DNA restriction fragments (7) and adducts (8), analysis of PCR products (9), DNA microarrays (10), mutation detection (11), enzymatic substrate monitoring (12), and various fluorescence resonance energy transfer (FRET)-based assays (11,13).…”
Section: Introductionmentioning
confidence: 99%