Detection and characterization of alpha-beta-T-cell clonality by denaturing gradient gel electrophoresis (DGGE).
Accumulation of T cells carrying identical T-cell receptors (TCR) is associated with a number of immunological and non-immunological diseases. Therefore, it is of interest to be able to analyze complex T-cell populations for the presence of clonally expanded subpopulations. Here, we describe a simple method combining reverse transcription (RT)-PCR and denaturing gradient gel electrophoresis (DGGE) for rapid detection and characterization of T-cell clonality. The detection of clonally expanded T cells by DGGE relies on the fact that clonal transcripts have no junctional diversity and therefore resolve at a fixed position in the gel, which is determined by their melting properties. For polyclonal populations with a high degree of junctional diversity, the different DNA molecules will resolve at different positions in the gel and together will be revealed as a smear. For each of the TCR beta-variable gene (BV) 1-24 families, cloned transcripts were amplified and shown to resolve as distinct bands in the denaturing gradient gel, whereas the analysis of polyclonal T-cell populations resulted in a smear in the gel. The present method might prove useful to test for clonotypic T-cells in a variety of pathological and physiological conditions and for monitoring T-cell responses in diagnostic and therapeutic settings.
Department of Tumor Cell Biology, Danish Cancer Society, Copenhagen, Denmark.
P
P
thor Straten
A
A
Barfoed
T
T
Seremet
I
I
Saeterdal
J
J
Zeuthen
P
P
Guldberg
D002478:Cells, Cultured; D002999:Clone Cells; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D009691:Nucleic Acid Denaturation; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D016693:Receptors, Antigen, T-Cell, alpha-beta