[Solid phase 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 possibility of using them in clinical biology has been considered for several years. The transfer from basic to applied research can now take place due to the new technology of non-radioactive probes (cold probes). Hybridization features the use of a probe nucleic acid molecule and of a target nucleic acid molecule. It leads to the formation of a double-stranded molecule, also called duplex, which can be detected with great sensitivity. For this to be done, the probe can be tagged with a variety of labels such as a radio-isotope or a non-isotopic marker which can be detected for its fluorescence, luminescence or enzyme activity or by immuno-reaction(s). Hybridization can be performed either in a liquid solution or on a solid support. In the first case, hybridization is generally followed by a separation step which makes use of solid phase to isolate the hybrid (duplex) formed. This separation can be achieved by biochemical means (adsorption chromatography, differential precipitation, electrophoresis, etc.) or by immunological means (affinity chromatography, immuno-capture). In the second case, the support can be used in different ways according to the assay format employed to perform hybridization. The target DNA can be immobilized by contacting the sample to be tested with the support: simple specimen deposit with a pipette (for low volumes), or by aspiration under vacuum or by (capillary or electrophoretic) transfer from an agarose gel. In the sandwich assay format, probe DNA is immobilized onto the support to specifically extract the target DNA from the medium under assay. Support to be employed for hybridization purpose can be selected from the group consisting of: nylon (N), nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membranes; N+NC mixed membranes; cellulose and its derivatives; polyoside supports (Sephacryl, Sepharose, etc.), latex particles, polystyrene microplates. The coupling procedures and capacity vary according to the type of support used.