Analysis of T cell receptor-gamma gene rearrangements by denaturing gradient gel electrophoresis of GC-clamped polymerase chain reaction products. Correlation with tumor-specific sequences.
We describe a modified denaturing gradient gel electrophoresis (DGGE) procedure with a 40-nucleotide GC clamp in the polymerase chain reaction to improve resolution in amplifying T cell receptor-gamma (TCR-gamma) rearrangements. DNA from 46 cases of lymphoblastic leukemia/lymphoma, 5T cell lines, 2 B cell lines, 7 normal lymphocytes, and 3 cases of Hodgkin's disease was amplified by polymerase chain reaction. In addition, 20 cases of paraffin-embedded T cell lymphomas and 5 cases of reactive hyperplasia were also studied. Clonal TCR-gamma rearrangements were identified on DGGE by the presence of a predominant band. Results obtained from 5 T cell lines and 12 lymphoblastic leukemia/lymphomas containing known TCR-gamma gene rearrangements revealed 100% concordance in detecting clonal rearrangements between DGGE and traditional Southern blot analysis. Of the remaining 34 lymphoblastic leukemia/lymphoma cases studied by DGGE alone, 30 were positive. DGGE analysis of 10 lymphoblastic leukemia/lymphoma cases with known group IV gamma to J gamma 1 or J gamma 2 rearrangement sequences confirmed that the electrophoretic migration was dependent on the tumor-specific rearranged TCR-gamma sequence. In addition, 17 of 20 cases of paraffin-embedded T cell lymphomas were positive by DGGE, 6 of which had the clonal population also identified in fresh tissue DNA. DGGE analysis of GC-clamped polymerase chain reaction products can provide a way to more accurately detect TCR-gamma clonality of lymphoid tumors and can be applied to archival tissues.
Hematopathology Section, National Cancer Institute, Bethesda, Maryland.
T C
TC
Greiner
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M
Raffeld
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C
Lutz
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F
Dick
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ES
Jaffe
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D015334:Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor; D006801:Humans; D007945:Leukemia, Lymphoid / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured