ANALYSIS OF REARRANGED T-CELL RECEPTOR BETA-CHAIN GENES BY POLYMERASECHAIN-REACTION (PCR) DNA-SEQUENCING AND AUTOMATED HIGH-RESOLUTION PCRFRAGMENT ANALYSIS

Polymerase chain reaction (PCR)-directed amplification and sequencing of rearranged immune genes for identification of clone-specific marker s are increasingly being used in acute lymphoblastic leukemia (ALL) an d non-Hodgkin's lymphoma (NHL) patients instead of the time consuming and labor intensive Southern analysis, In previous reports, no single common V beta and J beta sequence had been identified that allowed rel iable amplification of the majority of rearranged T-cell antigen recep tor (TCR)-beta V-D-J junctions at the DNA level because of the relativ ely large number of possible TCR-beta variable (V beta) and joining (J beta) gene segments involved in the rearrangement processes, In the p resent study we designed highly degenerate PCR primers directed agains t conserved sequences of the J beta genes, In combination with a previ ously published consensus V beta primer, these J beta primers specific ally amplify TCR-beta V-N(D)N-J junctions from genomic DNA. Using this approach we studied DNA extracted from biopsy material of nine patien ts with T-cell lymphoproliferative disorders, one c-ALL patient, and f ive patients with nonmalignant diseases, T-cell lines Molt 3, Jurkat, and HM 2 served as monoclonal controls, Individual PCR products were s equenced after cloning, The nucleotide sequences of 96 randomly chosen recombinant vectors were determined, In the polyclonal controls all a nalyzed clones differed in their TCR-beta V-N(D)N-J junctions, In the T-cell lines, in all of the T-cell malignancies, and in the c-ALL, mon oclonal PCR products could be identified by demonstration of clonally restricted V-N(D)N-J junctions, The PCR results were confirmed by auto mated fluorescence quantification and size determination of PCR produc ts after separation in a high-resolution polyacrylamide gel, The proce dure allows rapid and specific characterization of clonal TCR-P rearra ngements from genomic DNA and will significantly simplify current expe rimental approaches to identify and to quantitate malignant T cells du ring initial staging and follow-up of T-lineage NHL and ALL patients. (C) 1995 by The American Society of Hematology.