A SINGLE UNIVERSAL PRIMER FOR THE T-CELL RECEPTOR (TCR) VARIABLE GENES ENABLES ENZYMATIC AMPLIFICATION AND DIRECT SEQUENCING OF TCR-BETA CDNA OF VARIOUS T-CELL CLONES

We designed a primer for the PCR directed against a highly conserved sequence of the TCR Vbeta gene. The Vbeta-universal primer, in combination with a constant region-specific primer, enabled us to amplify TCRbeta cDNA of allo-HLA class-II-reactive T-cell clones by PCR without prior knowledge of their Vbeta sequences. The amplified TCR cDNA was purified by agarose gel electrophoresis and subjected to direct sequencing. In nine of ten T-cell clones analyzed, direct TCR sequencing gave readable sequence ladders, including two-thirds of Vbeta, junctional, and Jbeta regions. One T-cell clone gave an unreadable mixed-profile sequence ladder, indicating that this clone expressed more than one major TCRbeta transcript. Even in this case, however, it was possible to determine two different TCRbeta sequences separately using sequence primers specific to one of the 13 Jbeta segments deduced from the mixed ladder. Thus, direct sequencing utilizing the single Vbeta-universal primer enabled a simple, rapid, and reliable sequence determination of TCRbeta cDNA of all T-cell clones analyzed.