Discrimination of the V600E Mutation in BRAF by Rolling Circle Amplification and Förster Resonance Energy Transfer

Abstract : The 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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https://hal-normandie-univ.archives-ouvertes.fr/hal-02330858
Contributeur : Madeleine Roux-Merlin <>
Soumis le : jeudi 24 octobre 2019 - 10:51:51
Dernière modification le : samedi 26 octobre 2019 - 01:51:30

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Mariia Dekaliuk, Xue Qiu, Frédéric Troalen, Pierre Busson, Niko Hildebrandt. Discrimination of the V600E Mutation in BRAF by Rolling Circle Amplification and Förster Resonance Energy Transfer. ACS Sensors, American Chemical Society, 2019, ⟨10.1021/acssensors.9b01420⟩. ⟨hal-02330858⟩

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