title: Semireannealing, single-stranded conformational polymorphism: a novel and effective tool for the diagnosis of T-cell clonality.
abstract: 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.
authors.affiliation: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Giovanna Maria
authors.initials: GM
authors.lastname: Crisi
authors.forename: Janet Rettig
authors.initials: JR
authors.lastname: Emanuel
authors.forename: Chaline
authors.initials: C
authors.lastname: Johnson
authors.forename: Paul
authors.initials: P
authors.lastname: Crotty
authors.forename: Jose'
authors.initials: J
authors.lastname: Costa
authors.forename: Giovanni
authors.initials: G
authors.lastname: Tallini
mesh_terms: 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
chemical_list: D004273:DNA, Neoplasm; D016692:Receptors, Antigen, T-Cell, gamma-delta