In situ polymerase chain reaction technique revealed by flow cytometry as a tool for gene detection

Davide Gibellini, Giorgio Zauli, Maria Carla Re, Giuliano Furlini, Simona Lolli, Alessandra Bassini, Claudio Celeghini, Michele La Placa

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)252-258
Number of pages7
JournalAnalytical Biochemistry
Volume228
Issue number2
DOIs
Publication statusPublished - 1995

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

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