Analysis of the population of human T cell receptor gamma and delta chain variable region subfamilies by reverse dot blot hybridization.
We have developed a simple method to analyze the population of T cell receptor (TCR) gamma and delta chain variable (V) region subfamilies by the application of reverse dot blot hybridization, which was originally developed for the analysis of human HLA-DR polymorphism. The four oligonucleotides corresponding to each TCR-gamma V region subfamily and the six oligonucleotides to each TCR-delta V region gene were synthesized, and tailed with dTTP. The cDNA was amplified by ligation-mediated PCR in the presence of biotinylated deoxynucleotides. Hybridization between immobilized specific oligoprobes and biotinylated target DNA was nonradioactively detected by a reaction using alkaline phosphatase. The population of the V region subfamilies of TCR-gamma and -delta in PBL analyzed by reverse dot blot hybridization described here showed good correlations with the result of colony hybridization.
Tsuruta
Y
Y
Shionogi Research Laboratories, Shionogi & Co. Ltd., Osaka, Japan.
Yoshioka
T
T
Suzuki
R
R
Sakata
T
T
D001483:Base Sequence; D004247:DNA / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000032:analysis* / Q000235:genetics; D013601:T-Lymphocytes / Q000737:chemistry
D015345:Oligonucleotide Probes; D016692:Receptors, Antigen, T-Cell, gamma-delta; D012313:RNA; D004247:DNA