Document 0438
 DOCN  M9650438
 TI    PCR in situ: aspects which reduce amplification and generate
       false-positive results.
 DT    9605
 AU    Teo IA; Shaunak S; Department of Infectious Diseases, Royal Postgraduate
       Medical; School, London, UK.
 SO    Histochem J. 1995 Sep;27(9):660-9. Unique Identifier : AIDSLINE
       MED/96121506
 AB    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.
 DE    Autoradiography  Base Sequence  Blotting, Southern  Cell Line
       Cross-Linking Reagents  DNA Damage  DNA, Viral/ANALYSIS
       Electrophoresis, Agar Gel  False Positive Reactions  Formaldehyde  Gene
       Amplification  Histones/CHEMISTRY  Human  HIV
       Infections/DIAGNOSIS/VIROLOGY  HIV-1/GENETICS/ISOLATION & PURIF  In Situ
       Hybridization/*METHODS  Molecular Sequence Data  Polymerase Chain
       Reaction/*METHODS  Support, Non-U.S. Gov't  Tissue Fixation  JOURNAL
       ARTICLE

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