The influence of a DNA amplicon fragmentation on the efficiency of detecting a specific sequence by heterophase hybridization analysis was investigated. The nucleotide sequence was detected colorimetrically after biotinylation of an oligonucleotide probe immobilized on a solid carrier via limited elongation in complex with the sample DNA with the use of Taq polymerase. Two simple and reproducible techniques of amplicon fragmentation were proposed. The techniques are based on the introduction of apurinic/apyrimidinic sites in DNA and their subsequent thermal degradation. DNA was depurinated by a mild acidic treatment. Apyrimidinic sites were generated by treating a DNA fragment containing dUMP in place of some dTMP in various proportions with uracil-DNA glycosylase (UDG). The DNA sample treated by either method proved to be suitable for hybridization analysis with Taq polymerase without additional purification. The efficiency of hybridization analysis was higher with the fragmented than with the native DNA amplicon. DNA fragmentation makes it possible to use bridged oligonucleotides, having a lower hybridization ability, as highly selective hybridization probes.