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 journalArticle

20 Citations (Scopus)

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

Fingerprint

Digoxigenin
Flow cytometry
Polymerase chain reaction
Flow Cytometry
Genes
Polymerase Chain Reaction
HIV-1
Cells
DNA sequences
DNA
Fluorescein
Theoretical Models
Cell Count
Fluorescence
RNA
Cell Line
Antibodies

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

Cite this

Gibellini, D., Zauli, G., Re, M. C., Furlini, G., Lolli, S., Bassini, A., ... La Placa, M. (1995). In situ polymerase chain reaction technique revealed by flow cytometry as a tool for gene detection. Analytical Biochemistry, 228(2), 252-258. https://doi.org/10.1006/abio.1995.1347

In situ polymerase chain reaction technique revealed by flow cytometry as a tool for gene detection. / Gibellini, Davide; Zauli, Giorgio; Re, Maria Carla; Furlini, Giuliano; Lolli, Simona; Bassini, Alessandra; Celeghini, Claudio; La Placa, Michele.

In: Analytical Biochemistry, Vol. 228, No. 2, 1995, p. 252-258.

Research output: Contribution to journalArticle

Gibellini, D, Zauli, G, Re, MC, Furlini, G, Lolli, S, Bassini, A, Celeghini, C & La Placa, M 1995, 'In situ polymerase chain reaction technique revealed by flow cytometry as a tool for gene detection', Analytical Biochemistry, vol. 228, no. 2, pp. 252-258. https://doi.org/10.1006/abio.1995.1347
Gibellini, Davide ; Zauli, Giorgio ; Re, Maria Carla ; Furlini, Giuliano ; Lolli, Simona ; Bassini, Alessandra ; Celeghini, Claudio ; La Placa, Michele. / In situ polymerase chain reaction technique revealed by flow cytometry as a tool for gene detection. In: Analytical Biochemistry. 1995 ; Vol. 228, No. 2. pp. 252-258.
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