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 technique is still technically difficult and has not yet proved to be reliable or reproducible. We have now identified a number of factors which can contribute to the poor amplification of the target DNA and to the generation of false-positive signals. These factors include the effects of fixation, reagent abstraction, DNA degradation, DNA end-labelling and product diffusion. We present evidence to show that formaldehyde fixation cross-links histones to DNA and thus restricts the subsequent amplification of target sequences by PCR. End-labelling of DNA occurs when direct incorporation is used to detect amplified products and this gives rise to false-positive signals. Amplified products can also diffuse out of cells and into neighbouring cells which do not contain target sequences. They can undergo re-amplification within these cells giving rise to false-positive signals. We believe considerable caution should be exercised in the interpretation of results generated using PCR in situ.
Teo
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Department of Infectious Diseases, Royal Postgraduate Medical School, London, UK.
Shaunak
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D001345:Autoradiography; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D003432:Cross-Linking Reagents; D004249:DNA Damage; D004279:DNA, Viral / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D005189:False Positive Reactions; D005557:Formaldehyde; D005784:Gene Amplification; D015658:HIV Infections / Q000175:diagnosis / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification; D006657:Histones / Q000737:chemistry; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016707:Tissue Fixation
D003432:Cross-Linking Reagents; D004279:DNA, Viral; D006657:Histones; D005557:Formaldehyde