A single universal primer for the T-Cell receptor (TCR) variable genes enables enzymatic amplification and direct sequencing of TCRβ cDNA of various T-cell clones

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