Analysis of rearranged T-cell receptor beta-chain genes by polymerase chain reaction (PCR) DNA sequencing and automated high resolution PCR fragment analysis.
Polymerase chain reaction (PCR)-directed amplification and sequencing of rearranged immune genes for identification of clone-specific markers are increasingly being used in acute lymphoblastic leukemia (ALL) and 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 reliable amplification of the majority of rearranged T-cell antigen receptor (TCR)-beta V-D-J junctions at the DNA level because of the relatively large number of possible TCR-beta variable (V beta) and joining (J beta) gene segments involved in the rearrangement processes. In the present study we designed highly degenerate PCR primers directed against conserved sequences of the J beta genes. IN combination with a previously published consensus V beta primer, these J beta primers specifically amplify TCR- beta V-N(D)N-J junctions from genomic DNA. Using this approach we studied DNA extracted from biopsy material of nine patients with T-cell lymphoproliferative disorders, one c-ALL patient, and five patients with nonmalignant diseases. T-cell lines Molt 3, Jurkat, and HM 2 served as monoclonal controls. Individual PCR products were sequenced after cloning. The nucleotide sequences of 96 randomly chosen recombinant vectors were determined. In the polyclonal controls all analyzed 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, monoclonal PCR products could be identified by demonstration of clonally restricted V-N(D)N-J junctions. The PCR results were confirmed by automated fluorescence quantification and size determination of PCR products after separation in a high-resolution polyacrylamide gel. The procedure allows rapid and specific characterization of clonal TCR-beta rearrangements from genomic DNA and will significantly simplify current experimental approaches to identify and to quantitate malignant T cells during initial staging and follow-up of T-lineage NHL and ALL patients.
Kneba
M
M
Department of Internal Medicine, Georg-August University, Goettingen, Germany.
Bolz
I
I
Linke
B
B
Hiddemann
W
W
D001331:Automation; D001483:Base Sequence; D015139:Blotting, Southern; D004273:DNA, Neoplasm / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005470:Fluorometry; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; D006801:Humans; D007938:Leukemia / Q000235:genetics; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000235:genetics; D008199:Lymphadenitis / Q000235:genetics; D016399:Lymphoma, T-Cell / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods; D012689:Sequence Homology, Nucleic Acid; D013601:T-Lymphocytes / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D004274:DNA, Recombinant