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 s equence of the TCR Vbeta gene. The Vbeta-universal primer, in combinat ion with a constant region-specific primer, enabled us to amplify TCRb eta cDNA of allo-HLA class-II-reactive T-cell clones by PCR without pr ior knowledge of their Vbeta sequences. The amplified TCR cDNA was pur ified by agarose gel electrophoresis and subjected to direct sequencin g. In nine of ten T-cell clones analyzed, direct TCR sequencing gave r eadable sequence ladders, including two-thirds of Vbeta, junctional, a nd Jbeta regions. One T-cell clone gave an unreadable mixed-profile se quence ladder, indicating that this clone expressed more than one majo r TCRbeta transcript. Even in this case, however, it was possible to d etermine two different TCRbeta sequences separately using sequence pri mers specific to one of the 13 Jbeta segments deduced from the mixed l adder. Thus, direct sequencing utilizing the single Vbeta-universal pr imer enabled a simple, rapid, and reliable sequence determination of T CRbeta cDNA of all T-cell clones analyzed.