Clonotype-specific PCR amplification. Seminested PCR was carried out with clonotype-specific primers that, because they do not overlap the entire V-nDn-J junctions, enable the amplification of either identical or quasi-identical CDR3 sequences. Numbers identify PNH patients as from Table 1. Capital letters indicate healthy controls; *, patients in whom the clonotype had been previously identified by systematic cloning and sequencing; mw, molecular weight markers; rc, reagent control. (A) Clonotype-specific PCR for S4 clonotype. A specific S4 clonotype band is present in the 3 patients in whom it had been previously found by systematic cloning and sequencing (PNH 5, 7, 9; Table 2) and in 1 additional patient (PNH 10). The sequencing of the amplified products confirmed the identity of the sequence. (B) Clonotype-specific PCR for S5-like clonotype. A specific S5-like clonotype band is present in the 4 patients in whom it had been previously found by systematic cloning and sequencing (PNH 2, 5, 7, 9; Table 2) and in 4 additional patients (PNH 3, 14, 16, 20). The sequencing of the amplified products demonstrates the presence of both S5-identical and S5 quasi-identical sequences (Table 3). In addition, the PCR amplification in patient 19 yielded a longer PCR product whose sequence (CATSRGTSGRETQYFGP) revealed a CDR3 that uses the same TRBV-15 and TRBJ-2.5 of S5-like clonotypes but with a 6 nucleotide insertion that modifies significantly the nDn region. Therefore, this sequence has not been counted as belonging to the S5-like clonotype group.