AN EXPERIMENTALLY VALIDATED PANEL OF SUBFAMILY-SPECIFIC OLIGONUCLEOTIDE PRIMERS (V-ALPHA-1-W29/V-BETA-1-W24) FOR THE STUDY OF HUMAN T-CELL RECEPTOR VARIABLE V-GENE SEGMENT USAGE BY POLYMERASE CHAIN-REACTION

We report here the characterization of a series of T cell receptor (TcR) V(alpha) or V(beta) subfamily-specific oligonucleotide primers. Criteria that have guided the design of each oligonucleotide include appropriate thermodynamic parameters as well as differential base-pairing scores with related and unrelated target sequences. The specificity of the oligonucleotides for each V(alpha) or V(beta) subfamily was tested by polymerase chain reaction (PCR) on both a series of TcR encoding plasmid DNA and clonal T cell populations. Unexpected cross-reactivities were observed with plasmid cDNA sequences corresponding to unrelated subfamily gene segments. This led to the synthesis of additional series of oligonucleotides to obtain a relevant panel. A series of V(alpha)1-w29/V(beta)1-w24 TcR subfamily-specific oligonucleotides was eventually selected which generates little, if any, cross-reactivity. The use of C(alpha) or C(beta) primers for the amplification of internal positive control templates (i.e. C(beta) for the V(alpha) series and C(alpha) for the V(beta) series) has been tested in PCR performed with cDNA derived from peripheral blood lymphocytes; it was shown not to alter the amplification of the V subfamily-specific DNA fragments. This panel of oligonucleotides will be helpful in the study of TcR V gene segment usage and, thus, may lead to a better characterization of T cell response and, thus, may lead to a better characterization of T cell responses in physiological and pathological situations.