5-Fluorodeoxyuridine as an alternative to the synthesis of mixed hybridization probes for the detection of specific gene sequences

Synthetic complementary oligonucleotides are useful hybridization probes for the detection of mRNAs and genes encoding proteins for which only a partial amino acid sequence is known. Usually this involves the synthesis of mixtures of oligonucleotides complementary to all possible bases in degenerate positions of codons. As an alternative we have prepared and characterized a series of unique oligonucleotides containing a pyrimidine analog, 5-fluorodeoxyuridine (F). Thermodynamic parameters and the melting temperatures of hybrid duplexes containing A · F and G · F base pairs showed that they are considerably more stable than duplexes containing A · T and G · T base pairs. The stability of a duplex decreased linearly with the number of mismatches introduced at positions at least a codon apart. A 5-fluorodeoxyuridine-substituted oligonucleotide cDNA detects rat liver pyruvate carboxylase mRNA on a RNA gel blot with a dissociation temperature only 10°C below the measured melting temperature in solution. We suggest that the complexity of oligonucleotide cDNAs used for screening gene libraries can be reduced by the design of single hybridization probes containing the substituted bases - 5-fluorodeoxyuridine to pair with adenosine or guanosine, guanosine to pair with cytidine or thymidine, and deoxyinosine to pair with adenosine or cytidine at positions of codon degeneracy - and still retain near-maximum stability of hybrid duplexes.