In Situ Amplification Of Target Sequence

6.1.1. Primed In Situ Labeling The technique of primed in situ (PRINS) labeling described originally by Koch in 1992 (55) overcomes the occasional problems with cross-hybridization of CEP probes resulting from the homology of alphoid repetitive sequences among different chromosomes (e.g., chromosomes 13 and 21). A specific primer is used for the region of interest and it is elongated on 3' end using labeled nucleotides and a thermostable Taq DNA polymerase. Repeated cycles of primed in situ synthesis over the target would enhance the signal in a process called "cycling PRINS." This method is considered to be rapid, and more sensitive than ISH or FISH. It is possible to apply PRINS to interphase nuclei and, thus, the possibility of using archival tissue materials demonstrate its clinical implications.

6.1.2. In Situ PCR Polymerase chain reaction amplification of a low copy number of viruses was first described by Haase in 1990 (56). It is now possible to detect as low as one copy number of a nucleic acid sequence in a single cell using this technique of in situ PCR (54). For PRINS and cycling PRINS or in situ PCR, again as seen with ISH/FISH, crosslink-ing fixatives like formalin are recommended because they retain the amplification product at the target sequence subcel-lular location, whereas the precipitating fixatives like Bouin's fluid tend to diffuse the product. Using specific primer(s) and Taq polymerase, an in situ amplification could be achieved either in a suspension of fixed cells in a regular thermocycler heating block or carried out on a special heating block in cells/tissues fixed on a glass slide. The detection of the amplified product then is carried out on a glass slide using a directly labeled FISH probe or a FISH signal reporting system if hapt-enized nucleotides were used in the PCR reaction. Despite initial enthusiasm, the technique at the end of the decade after its inception is still dwindling in use from routine clinical application because of the high false-positive and false-negative rates.

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