Abstract
Forced intercalation probes (FIT-probes) are peptide nucleic acid-based probes in which the thiazole orange dye replaces a canonical nucleobase. FIT-probes are used in homogenous DNA detection. The analysis is based on sequence-specific binding of the FIT-probe with DNA. Binding of the FIT-probe places thiazole orange in the interior of the formed duplex. The intercalation of thiazole orange between nucleobases of the formed probe-target duplex restricts the torsional flexibility of the two heterocyclic ring systems. As a result, FIT probes show strong enhancements of fluorescence upon hybridization. A remarkable attenuation of fluorescence is observed when forcing thiazole orange to intercalate next to a mismatched base pair. This base specificity of fluorescence signaling, which adds to the specificity of probe-target recognition, allows the detection of single base mutations even at non-stringent hybridization conditions.
The performance of FIT-probes in real-time PCR is demonstrated in an assay for the SNPtyping of human H-ras. FIT-probe was added at the start of a real-time amplification containing the wild-type (G,G)-allele, mutant (T,T)-allele or heterozygous (G,T)-allele of the human H-ras gene. The identity of the target DNA is determined in real time due to significant differences in signal intensities.
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Socher, E., Seitz, O. (2008). FIT-Probes in Real-Time PCR. In: Marx, A., Seitz, O. (eds) Molecular Beacons: Signalling Nucleic Acid Probes, Methods, and Protocols. Methods in Molecular Biology, vol 429. Humana Press. https://doi.org/10.1007/978-1-60327-040-3_13
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DOI: https://doi.org/10.1007/978-1-60327-040-3_13
Publisher Name: Humana Press
Print ISBN: 978-1-58829-700-6
Online ISBN: 978-1-60327-040-3
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