Polymerase chain reaction in situ: an appraisal of an emerging technique.
Polymerase chain reaction (PCR) in situ is a new technique which promises to enhance considerably our ability to detect a few copies of target nucleic acid sequences in fixed tissues and cells. It has an enormous potential for application in diagnostic histopathology of viral diseases and in the study of gene expression. PCR in situ is, however, technically difficult, and amplification of the target DNA is only 30-300 fold. In this article we present an overview of PCR in situ techniques 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 inhibitory effects of cross-linking of histones to DNA or PCR amplification, (2) abstraction of PCR reagents by tissue-bonding agents which are used to coat glass slides, (3) poor denaturation of target DNA and subsequent DNA renaturation due to extensive cross-linking of histones to DNA, or because of incorrect temperature regulation of thermal cyclers, (4) false-positive results which arise from end-labelling of DNA strand breaks by Taq polymerase, and (5) diffusion of PCR products into and out of cells leading to false-positive results. We present some of the approaches that have been used to overcome some of these difficulties and suggest new avenues for investigation to improve this technique further.
Teo
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Department of Infectious Diseases, Royal Postgraduate Medical School, London, UK.
Shaunak
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D000818:Animals; D004247:DNA / Q000032:analysis; D005189:False Positive Reactions; D015870:Gene Expression; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D016707:Tissue Fixation
D012333:RNA, Messenger; D004247:DNA