title: 32P-incorporation PCR for the detection of rearrangements at the TCR-gamma locus.
abstract: We have adapted and developed a PCR (polymerase chain reaction)-based technique for the T-cell receptor (TCR)-gamma chain gene, which has subsequently been used for routine diagnosis. Variable-region oligonucleotide primers were chosen from subgroups I and II, and the joining region primer was from the J2 segment. The primers were used to perform a 32P-incorporation PCR, and the products were then separated on an 8% denaturing polyacrylamide gel. In our hands, this technique is more reliable than cold methods, when separation is performed on either agarose or nondenaturing polyacrylamide. The radioactive technique was used to look at 102 T-cell proliferations, of which eight of eight T-acute lymphoblastic leukemia (ALL), 24 of 34 T-non-Hodgkin's leukemia (NHL), and 35 of 60 large granular lymphocyte (LGL) expansions were clonal. Of 122 B-cell proliferations investigated, including 72 cases of B-cell lineage ALL, 36 demonstrated a T-cell rearrangement (33 ALLs and three myelomas). Samples from nonlymphoid tumors were tested and produced a normal distribution ladder of PCR products after autoradiography, a pattern also observed with antenatal and preoperative patients. The radiolabel-incorporation method detected an abnormal pattern of a ladder with prominent dark bands in 29 of 122 B-cell and 27 of 102 T-cell cases and in 0 of 49 of the nonlymphoid and normal samples. The abnormal banding patterns obtained in a proportion of the B- and T-cell cases was not readily discernible by nondenaturing-acrylamide or agarose-separation methods.
authors.affiliation: Department of Molecular Haematology, Leeds General Infirmary, England.
authors.forename: M A
authors.initials: MA
authors.lastname: Short
authors.forename: P A
authors.initials: PA
authors.lastname: Evans
authors.forename: C R
authors.initials: CR
authors.lastname: Shiach
authors.forename: A
authors.initials: A
authors.lastname: Jack
authors.forename: S
authors.initials: S
authors.lastname: Richards
authors.forename: G J
authors.initials: GJ
authors.lastname: Morgan
mesh_terms: D015139:Blotting, Southern; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015329:Gene Rearrangement, T-Lymphocyte; D006801:Humans; D008232:Lymphoproliferative Disorders / Q000235:genetics* / Q000473:pathology; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics*; D013696:Temperature
chemical_list: D010761:Phosphorus Radioisotopes; D016692:Receptors, Antigen, T-Cell, gamma-delta