Identification of predominant T-cell receptor rearrangements by temperature-gradient gel electrophoresis and automated DNA sequencing.
To assess the diversity of T-cell receptor (TCR) gene rearrangements in uncloned lymphocytes we used a three-stage strategy that allows the detection of a restricted TCR repertoire and the identification of the predominant, rearranged sequence(s). We have analyzed in parallel T cells obtained from a renal cell carcinoma infiltrate that specifically lyse the autologous tumor after in vitro culture and T cells from autologous peripheral blood. First, DNA amplification by the polymerase chain reaction (PCR) was performed with a number of oligonucleotide primers specific for several TCR V alpha gene families. All V alpha primers displayed specific amplification products in the peripheral blood, while a restricted TCR repertoire was present in the tumor-infiltrating lymphocytes. Subsequently, positively amplified PCR products were run in a temperature-gradient gel electrophoresis. A limited number of bands corresponding to predominant homo- and heteroduplexes was only found in the tumor-infiltrating lymphocytes. The presence of a low number of rearranged TCR sequences in these samples was confirmed by automated single-stranded DNA sequencing using a single fluorescent dye. These results support the broad application of this strategy for targeted sequencing of those PCR products carrying predominant DNA templates without previous DNA cloning.
Segurado
O G
OG
Institute of Immunology, University of Munich, Germany.
Schendel
D J
DJ
D001322:Autoanalysis; D001483:Base Sequence; D002292:Carcinoma, Renal Cell / Q000235:genetics; D004247:DNA / Q000737:chemistry; D004586:Electrophoresis; D015329:Gene Rearrangement, T-Lymphocyte / Q000235:genetics*; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013601:T-Lymphocytes / Q000737:chemistry; D013696:Temperature; D014407:Tumor Cells, Cultured
D004247:DNA