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The Histochemical Journal

, Volume 27, Issue 9, pp 660–669 | Cite as

PCRin situ: aspects which reduce amplification and generate false-positive results

  • I. A. Teo
  • S. Shaunak
Papers
  • 25 Downloads

Summary

PCRin 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 PCRin situ.

Keywords

Formaldehyde Nucleic Acid Target Sequence Neighbouring Cell Considerable Effort 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Chapman & Hall 1995

Authors and Affiliations

  • I. A. Teo
    • 1
  • S. Shaunak
    • 1
  1. 1.Human Retrovirus Group, Department of Infectious DiseasesRoyal Postgraduate Medical SchoolLondonUK

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