POLYMERASE CHAIN-REACTION IN-SITU - AN APPRAISAL OF AN EMERGING TECHNIQUE

Polymerase chain reaction (PCR) in situ is a new technique which promi ses to enhance considerably our ability to detect a few copies of targ et nucleic acid sequences in fixed tissues and cells. It has an enormo us potential for application in diagnostic histopathology of viral dis eases and in the study of gene expression. PCR in situ is, however, te chnically difficult, and amplification of the target DNA is only 30-30 0 fold. In this article we present an overview of PCR in situ techniqu es used to amplify both DNA and RNA targets (RT-PCR in situ). We also identify problems which can reduce the efficiency of the technique or which can give rise to false-positive results. They include (1) the in hibitory effects of cross-linking of histones to DNA or PCR amplificat ion, (2) abstraction of PCR reagents by tissue-bonding agents which ar e used to coat glass slides, (3) poor denaturation of target DNA and s ubsequent DNA renaturation due to extensive cross-linking of histones to DNA, or because of incorrect temperature regulation of thermal cycl ers, (4) false-positive results which arise from end-labelling of DNA strand breaks by Tag polymerase, and (5) diffusion of PCR products int o and out of cells leading to false-positive results. We present some of the approaches that have been used to overcome some of these diffic ulties and suggest new avenues for investigation to improve this techn ique further.