Semireannealing, single-stranded conformational polymorphism: a novel and effective tool for the diagnosis of T-cell...

Single-stranded conformational polymorphism (SSCP) is often used for the diagnosis of T-cell clonality in lymphoproliferative disorders. We introduce a semireannealing SSCP (SR-SSCP) protocol that is rapid, reproducible, and effective. By denaturing and reannealing the polymerase chain reaction (PCR) product before high-resolution polyacrylamide gel electrophoresis, it is possible to generate a diagnostic fingerprint for each case with clonal T-cell receptor-gamma (TCR-gamma) gene rearrangement detected after PCR with TCR-gamma specific consensus primers. Discrete and distinct denatured single-stranded DNA band profiles characterize the rearranged TCR-gamma clones. In the same gel, the clone size may be estimated in the reannealed double-stranded PCR DNA and can be assessed down to the 2% clonal T-cell population level. Eighty-four cases, including 37 T-cell neoplasms, 29 B-cell neoplasms, and 18 reactive lymph node samples were analyzed by SR-SSCP. Clonal TCR-gamma rearrangement was diagnosed in 32 out of 37 T-cell neoplasms but in none of the B-cell tumors or reactive lymph node samples corresponding to sensitivity and specificity of 86.5% and 100%, respectively. We compare the results of SR-SSCP to those obtained by capillary electrophoresis and direct sequence analysis with 100% correlation. This novel method is applicable to any system for identification and quantitation of microheterogeneity in PCR products.