Identification of predominant T-cell receptor rearrangements by temperature-gradient gel electrophoresis and automated DNA sequencing

✍ Scribed by Dr. Oscar G. Segurado; Dolores J. Schendel

• Publisher: John Wiley and Sons
• Year: 1993
• Tongue: English
• Weight: 772 KB
• Volume: 14
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/elps.11501401117

✦ Synopsis

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 a gene families. All Vc 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 turnor-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.