Future Directions In Fishbased Assays

Currently, the major limitations of FISH is that it can only detect large numerical and structural changes and that it lacks the sensitivity of detection of a single nucleotide change as offered by PCR/sequencing. The development of so-called "padlock probes" offers the luxury of detecting single-basepair mismatch (88). These are small oligonucelotide haptenized probes having extensions of oligonucleotides about 20 bp long at 3' and 5' ends, which are selected carefully to be juxtaposed upon their hybridization to the sequence of interest. The additional ligation reaction closes the ends of the padlock and attaches the probe covalently to the target. Because it requires two hybridization events over relatively very small stretches of targets, even a single-basepair mismatch hampers the hybridization of padlock probes. Perhaps their combination with amplification systems like fluorescent tyramides would improve their sensitivity further. Along with padlock probes, the amplification systems like PRINS, in situ PCR, and tyra-mide systems are expected to gain increasing prominence in FISH-based assays in the future. As mentioned earlier, clinical applications of FISH are already on the forefronts of hematol-ogy. However, what is exciting is the progress in solid tumor cytogenetics with mixtures of multiple probes and diverse sampling methods that could be expected to bring significant advances in solid tumor management (for review, see ref. 89). A great promise is also felt in the utility of FISH- based assays for cancer risk screening, which will be ascertained in the next few years. Moreover, the technique of genomic microarray is in the wings to make an entry onto a routine clinical stage.

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