Document 0028
 DOCN  M9650028
 TI    In situ polymerase chain reaction technique revealed by flow cytometry
       as a tool for gene detection.
 DT    9605
 AU    Gibellini D; Zauli G; Re MC; Furlini G; Lolli S; Bassini A; Celeghini C;
       La Placa M; Institute of Microbiology, University of Bologna, Italy.
 SO    Anal Biochem. 1995 Jul 1;228(2):252-8. Unique Identifier : AIDSLINE
       MED/96171045
 AB    We report a methodology for detecting specific DNA sequences directly
       inside cells, combining in situ PCR and flow cytometry. This technique
       is based on in situ PCR performed in the presence of digoxigenin-labeled
       dUTP to obtain a digoxigenin-labeled amplicon, which is then revealed by
       an anti-digoxigenin polyclonal antibody directly conjugated to
       fluorescein. Fluorescence intensity is next evaluated by flow cytometry.
       Our experimental models were represented by the lymphoblastoid cell
       lines 8E5LAV, carrying an integrated HIV-1 DNA proviral copy per cell,
       and A.301, infected in vitro with HIV-1 (strain IIIB). The technique is
       described in detail with particular attention to the optimization of
       critical fixation and permeabilization steps. This method allows not
       only the detection but also an accurate quantification of the number of
       positive cells in a background of negative cells. Moreover, it has the
       potentiality to develop into a multiparametric method for the
       simultaneous study of specific DNA or RNA sequences and surface or
       intracellular markers.
 DE    Base Sequence  Cell Line  Cell Membrane Permeability  Cloning, Molecular
       Digoxigenin  DNA, Viral/*ANALYSIS  *Flow Cytometry  Gene Products,
       gag/ANALYSIS  HIV-1/*GENETICS  In Situ Hybridization
       Lymphocytes/VIROLOGY  Models, Biological  Molecular Sequence Data
       Polymerase Chain Reaction/*METHODS  Stem Cells/VIROLOGY  Support,
       Non-U.S. Gov't  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
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