PCR IN-SITU - ASPECTS WHICH REDUCE AMPLIFICATION AND GENERATE FALSE-POSITIVE RESULTS

PCR in situ promises the ability to amplify and detect very low levels of target nucleic acid in tissues. Despite considerable effort, the t echnique is still technically difficult and has not yet proved to be r eliable or reproducible. We have now identified a number of factors wh ich can contribute to the poor amplification of the target DNA and to the generation of false-positive signals. These factors include the ef fects of fixation; reagent abstraction, DNA degradation, DNA end-label ling and product diffusion. We present evidence to show that formaldeh yde fixation cross-links histones to DNA and thus restricts the subseq uent amplification of target sequences by PCR. End-labelling of DNA oc curs when direct incorporation is used to detect amplified products an d this gives rise to false-positive signals. Amplified products can al so diffuse out of cells and into neighbouring cells which do not conta in target sequences. They can undergo re-amplification within these ce lls giving rise to false-positive signals. We believe considerable cau tion should be exercised in the interpretation of results generated us ing PCR in situ.