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 V beta gene. The V beta-universal primer, in combination with a constant region-specific primer, enabled us to amplify TCR beta cDNA of allo-HLA class-II-reactive T-cell clones by PCR without prior knowledge of their V beta 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 V beta, junctional, and J beta regions. One T-cell clone gave an unreadable mixed-profile sequence ladder, indicating that this clone expressed more than one major TCR beta transcript. Even in this case, however, it was possible to determine two different TCR beta sequences separately using sequence primers specific to one of the 13 J beta segments deduced from the mixed ladder. Thus, direct sequencing utilizing the single V beta-universal primer enabled a simple, rapid, and reliable sequence determination of TCR beta cDNA of all T-cell clones analyzed.
Obata
F
F
Laboratory of Immunology, Kitasato University School of Medicine, Sagamihara, Japan.
Tsunoda
M
M
Ito
K
K
Ito
I
I
Kaneko
T
T
Pawelec
G
G
Kashiwagi
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D002999:Clone Cells / Q000737:chemistry; D004247:DNA / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid; D013601:T-Lymphocytes / Q000737:chemistry*
D009838:Oligodeoxyribonucleotides; D016693:Receptors, Antigen, T-Cell, alpha-beta; D004247:DNA