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Discrimination of the V600E Mutation in BRAF by Rolling Circle Amplification and Förster Resonance Energy Transfer
journal contribution
posted on 2019-10-15, 17:37 authored by Mariia Dekaliuk, Xue Qiu, Frédéric Troalen, Pierre Busson, Niko HildebrandtThe
quantification of very low concentrations of circulating tumor
DNA (ctDNA) biomarkers from liquid biopsies has become an important
requirement for clinical diagnostics and personalized medicine. In
particular, the simultaneous detection of wild-type (WT) dsDNA and
their cancer-related counterparts presenting single-point mutations
with simple, sensitive, specific, and reproducible technologies is
paramount for ctDNA assays in clinical practice. Here, we present
the development and evaluation of an amplified dsDNA assay based on
a combination of isothermal rolling circle amplification (RCA) and
time-gated Förster resonance energy transfer (TG-FRET) between
a Tb donor and two dye (Cy3.5 and Cy5.5) acceptors. The RCA–FRET
assay is free of washing and separation steps and can quantify both
WT and mutated (MT) (V600E) dsDNA in the BRAF gene from a single sample in the 75 fM to 4.5 pM (4.5
× 105 to 2.7 × 107 copies) concentration
range. This assay includes all steps from denaturation of the dsDNA
targets to the final duplexed quantification of WT and MT targets.
High assay performance at different dsDNA sequence lengths and high
target specificity even in the presence of a large excess of nonspecific
cell-free DNA from human plasma samples demonstrated the applicability
to clinical samples. The RCA–FRET single-point mutation sensor
has the potential to become an important complementary technique for
analyzing liquid biopsies in advanced cancer diagnostics.
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High assay performance75 fMdsDNA assayWTCy 3.5concentration rangebiopsyTG-FRETV 600Ecircle amplificationTb donorRolling Circle AmplificationctDNA assaysplasma samplesseparation stepsMT targetsFodsDNA targetsdsDNA sequence lengthsV 600E Mutationtumor DNAtarget specificitysingle-point mutationscancer diagnosticsRCABRAF geneduplexed quantification
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