Improved Polymerase Chain Reaction Detection of Clonally Rearranged T-Cell Receptor β Chain Genes


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Abstract

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 p chain (TcRβ) 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 Jβ segment involved in a clonal rearrangement. A Jβ-primer was used for the second PCR (Jβ-specific PCR), recognizing the Jβ segment identified in the screening PCR in combination with a consensus Vβ primer. This PCR generated prominent and short amplificates suitable for direct sequence analysis because of their low background. Using this approach, clonal TcRβ 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 TcRβ 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 TcRβ 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 Jβ-specific PCR with high sensitivity and specificity.

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