TCR beta PCR from crude preparations for restriction digest or sequencing.
T cell specificity is determined by the combinatorial association of specific variable (V), diversity (D), and junctional (J) regions. Clones of T cells (clonality) can occur, in the blood or in tissue, after proliferation of activated T cells. Determining clonality in mutation assays is necessary to distinguish between mutants and mutational events. We have developed a novel approach to determine clonality among T cell isolates, using restriction digests of PCR-amplified cDNA of the T cell receptor beta gene. The T cell receptor beta gene was PCR-amplified by use of a consensus primer, beginning from a cell pellet of 2,000-5,000 cells or from extracted RNA. This TCR (T cell receptor) beta chain PCR product can also be directly sequenced, allowing simple and easy identification of Vbeta and CDR3 sequence from a small number of cells. The utility of this method is demonstrated by PCR, restriction digest, and sequencing of the TCR beta cDNA from eight T cell clones isolated from 2 individuals. A clone of three identical isolates (one 3-mer) and a clone of two identical isolates (one 2-mer) were determined from restriction digests using two different enzymes. This new method is an easier and more rapid way of determining clonality than traditional methods, e.g., Southern blotting.
Clark
L S
LS
Vermont Cancer Center Genetics Laboratory, University of Vermont, Burlington, 05401, USA.
Nicklas
J A
JA
D001483:Base Sequence; D002999:Clone Cells; D004247:DNA / Q000302:isolation & purification*; D018076:DNA, Complementary / Q000235:genetics; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013601:T-Lymphocytes / Q000737:chemistry*
D018076:DNA, Complementary; D016693:Receptors, Antigen, T-Cell, alpha-beta; D004247:DNA