Semireannealing, single-stranded conformational polymorphism: a novel and effective tool for the diagnosis of T-cell clonality.
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.
Crisi
Giovanna Maria
GM
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Emanuel
Janet Rettig
JR
Johnson
Chaline
C
Crotty
Paul
P
Costa
Jose'
J
Tallini
Giovanni
G
D001483:Base Sequence; D002999:Clone Cells / Q000473:pathology; D004273:DNA, Neoplasm / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D015321:Gene Rearrangement; D006801:Humans; D008198:Lymph Nodes / Q000473:pathology; D016393:Lymphoma, B-Cell / Q000235:genetics / Q000473:pathology; D016399:Lymphoma, T-Cell / Q000235:genetics / Q000473:pathology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D019310:Pseudolymphoma / Q000235:genetics / Q000473:pathology; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D004273:DNA, Neoplasm; D016692:Receptors, Antigen, T-Cell, gamma-delta