TCR-BETA PCR FROM CRUDE PREPARATIONS FOR RESTRICTION DIGEST OR SEQUENCING

T cell specificity is determined by the combinatorial association of s pecific variable (V), diversity (D), and junctional (J) regions. Clone s of T cells (clonality) can occur, in the blood or in tissue, after p roliferation of activated T cells. Determining clonality in mutation a ssays is necessary to distinguish between mutants and mutational event s. We have developed a novel approach to determine clonality among T c ell isolates, using restriction digests of PCR-amplified cDNA of the T cell receptor beta gene. The T cell receptor beta gene was PCR-amplif ied by use of a consensus primer, beginning from a cell pellet of 2,00 0-5,000 cells or from extracted RNA. This TCR (T cell receptor) beta c hain PCR product can also be directly sequenced, allowing simple and e asy identification of V beta 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 i solated from 2 individuals. A clone of three identical isolates (one 3 -mer) and a clone of two identical isolates (one 2-mer) were determine d from restriction digests using two different enzymes. This new metho d is an easier and more rapid way of determining clonality than tradit ional methods, e.g., Southern blotting. (C) 1996 Wiley-Liss, Inc.