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 Va gene families. All Vα 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.