Simple chemical synthesis and hybridization properties of non-radioactive DNA probes

A simple chemical method for the synthesis of non-radioactive DNA probes is described: triazolyl-containing sequences were built by incorporation of 4-triazolylpyrimidin-2-ones instead of cytidines during oligodeoxyribonucleotide synthesis. The activating triazolyl groups were then displaced by a diamine which was further derivatized by a label, such as biotin. Synthesized DNA probes were oligonucleotides complementary to a cloned human antithrombin III DNA sequence. These probes, containing the same label at different positions of the sequence, were hybridized to their target DNA immobilized on nitrocellulose. Their hybridization specificity and stability were studied. Hybrid detection was performed either colorimetrically by the streptavidin-alkaline phosphatase-based system or by autoradiography after 5'-32P labeling of the probes: 15 fmol (0.05 microgram) of complementary sequence could be visualized in the two cases.