Target nucleic acid fragments separated by gel electrophoresis
C Sandwich hybridization
Target fragments denatured to single strand (not visible on membrane)
Hybridization of probe with specific target fragment
Hybridization between target and capture probe
Detection of probe—target fragment duplex by reporter molecule
Target nucleic acid \ ^
■ Capture probe
Hybridization between labeled probe and target nucleic acid detected via signal produced by reporter molecule subjected to an electrical field that forces them to migrate through an agarose matrix. Because fragments of different sizes migrate through the porous agarose at different rates, they can be separated over the time they are exposed to the electrical field. When electrophoresis is complete, the nucleic acid fragments are stained with the fluorescent dye ethidium bromide so that fragment "banding patterns" may be visualized on exposure of the gel to ultraviolet (UV) light. For Southern hybridization, the target nucleic acid bands are transferred to a membrane that is then exposed to probe nucleic acid. This method allows the determination of which specific target nucleic acid fragment is carrying the base sequence of interest, but the labor intensity of the procedure precludes its common use in most diagnostic settings.
With sandwich hybridizations two probes are used. One probe is attached to the solid support, is not labeled, and via hybridization "captures" the target nucleic acid from the sample to be tested. The presence of this duplex is then detected using a labeled second probe that is specific for another portion of the target sequence (see Figure 8-4, C). Sandwiching the target between two probes decreases nonspecific reactions but requires a greater number of processing and washing steps. For such formats, plastic microtiter wells coated with probes have replaced filters as the solid support material, thereby facilitating these multiple-step
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