Forced intercalation probes (FIT Probes): thiazole orange as a fluorescent base in peptide nucleic acids for homogeneous single-nucleotide-polymorphism detection.
Fluorescent base analogues in DNA are versatile probes of nucleic acid-nucleic acid and nucleic acid-protein interactions. New peptide nucleic acid (PNA) based probes are described in which the intercalator dye thiazole orange (TO) serves as a base surrogate. The investigation of six TO derivatives revealed that the linker length and the conjugation site decided whether a base surrogate conveys sequence-selective DNA binding and whether fluorescence is increased or decreased upon single-mismatched hybridization. One TO derivative conferred universal PNA-DNA base pairing while maintaining duplex stability and hybridization selectivity. TO fluorescence increased up to 26-fold upon hybridization. In contrast to most other probes, in which fluorescence is invariant once hybridization had occurred, the emission of TO-containing PNA probes is attenuated when forced to intercalate next to a mismatched base pair. The specificity of DNA detection is therefore not limited by the selectivity of probe-target binding and a DNA target can be distinguished from its single-base mutant under nonstringent hybridization conditions. This property should be of advantage for real-time quantitative PCR and nucleic acid detection within living cells.
Köhler
Olaf
O
Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany.
Jarikote
Dilip Venkatrao
DV
Seitz
Oliver
O
D001483:Base Sequence; D052160:Benzothiazoles; D004247:DNA / Q000378:metabolism; D015342:DNA Probes / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008969:Molecular Sequence Data; D020135:Peptide Nucleic Acids / Q000032:analysis / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide; D011804:Quinolines; D013844:Thiazoles / Q000737:chemistry*
D052160:Benzothiazoles; D015342:DNA Probes; D005456:Fluorescent Dyes; D020135:Peptide Nucleic Acids; D011804:Quinolines; D013844:Thiazoles; C051000:thiazole orange; D004247:DNA