In situ polymerase chain reaction technique revealed by flow cytometry as a tool for gene detection.
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.
Gibellini
D
D
Institute of Microbiology, University of Bologna, Italy.
Zauli
G
G
Re
M C
MC
Furlini
G
G
Lolli
S
S
Bassini
A
A
Celeghini
C
C
La Placa
M
M
D001483:Base Sequence; D002460:Cell Line; D002463:Cell Membrane Permeability; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000032:analysis*; D004076:Digoxigenin; D005434:Flow Cytometry; D015683:Gene Products, gag / Q000032:analysis; D015497:HIV-1 / Q000235:genetics*; D017403:In Situ Hybridization; D008214:Lymphocytes / Q000821:virology; D008954:Models, Biological; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D013234:Stem Cells / Q000821:virology
D004279:DNA, Viral; D015683:Gene Products, gag; D004076:Digoxigenin