Improved polymerase chain reaction detection of clonally rearranged T-cell receptor beta chain genes.
A new method for the detection of all known possible rearrangements at the variable (V), diversity (D), and joining (J) segments of the T-cell receptor beta chain (TcR beta) gene in tissue DNA extracts is described that involves two polymerase chain reactions (PCRs). The first PCR round (screening PCR) allowed the identification of the J beta segment involved in a clonal rearrangement. A J beta-primer was used for the second PCR (J beta-specific PCR), recognizing the J beta segment identified in the screening PCR in combination with a consensus V beta primer. This PCR generated prominent and short amplificates suitable for direct sequence analysis because of their low background. Using this approach, clonal TcR beta gene rearrangements were able to be demonstrated in all T-cell lines (n = 7) and in all peripheral T-cell lymphomas (n = 33) analyzed. No clonal TcR beta gene rearrangements were found in any of the normal tissues studied nor in any B-cell non-Hodgkin lymphomas. This method is applicable to DNA from fresh frozen tissues, and, after the TcR beta rearrangement of a patient's malignant T-cell clone has been identified by the screening PCR, DNA can also be detected in follow-up formalin-fixed paraffin-embedded samples by the J beta-specific PCR with high sensitivity and specificity.
Zemlin
M
M
Konsultations- und Referenzzentrum für Lymphknoten- und Hämatopathologie, Klinikum Benjamin Franklin, Free University Berlin, Germany.
Hummel
M
M
Anagnostopoulos
I
I
Stein
H
H
D002999:Clone Cells; D004247:DNA / Q000032:analysis*; D017931:DNA Primers / Q000737:chemistry; D015333:Gene Rearrangement, beta-Chain T-Cell Antigen Receptor / Q000235:genetics*; D019674:Genes, T-Cell Receptor beta / Q000235:genetics; D006801:Humans; D007144:Immunoglobulin J-Chains / Q000235:genetics; D016393:Lymphoma, B-Cell / Q000235:genetics; D016410:Lymphoma, T-Cell, Cutaneous / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D012751:Sezary Syndrome / Q000235:genetics; D013601:T-Lymphocytes; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D007144:Immunoglobulin J-Chains; D016693:Receptors, Antigen, T-Cell, alpha-beta; D004247:DNA