[Nonradioactive molecular hybridization]

Abstract

Nucleic acid hybridization techniques have been used for several decades in basic research to isolate genes, to determine their structure or analyse their mechanisms. The new technology of non-radioactive probes (cold probes) allows the routine use of this method outside of the specialized molecular biology laboratory. Hybridization makes use of a nucleic acid probe to detect a complementary nucleic acid target present in biological fluid or in a biopsy tissue. Hybridization leads to the formation of a double-stranded molecule called hybrid or duplex, which can be detected with a great sensitivity by using high-energy radioisotope. However, the use of radioisotope labeled probes is limited by the short half-life of the isotope, radiolysis of the probe and the radioactive hazards. In order to overcome autoradiography delay and the drawbacks of techniques employing radioisotopes, various non-radioactive labels have been proposed. Labeling and detection systems are currently designed in two ways: The label molecule can be attached directly to the DNA probe (direct labelling) or it can be attached to a molecule which binds either to a modified probe or specifically to the duplex (indirect labeling). Various substances has been used to label directly a nucleic acid probe. The assay detection limit depends largely on the detection limit of the label, therefore, probe assay based on label which provides signal amplification (e.g., enzymes) is likely to be more sensitive than an assay using a label which provides only a single signal per molecule (e.g., fluorochromes). In the indirect labeling, the probe cannot be detected alone; rather, it requires the addition of a detection system.(ABSTRACT TRUNCATED AT 250 WORDS)