ISSN:
0173-0835
Keywords:
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
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.
Additional Material:
4 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.11501401117
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