Single-strand conformation polymorphisms can be used to detect T cell receptor gene rearrangements: an application to the in vivo hprt mutation assay.
Epidemiologic application of the human in vivo hypoxanthine-guanine phosphoribosyltransferase (hprt) mutation assay requires screening of mutant colonies to differentiate independent from clonal origin. Previously, sibship was defined by Southern blot analysis of T cell receptor gene rearrangements. We report here a more expedient method to determine these rearrangements utilizing the polymerase chain reaction (PCR) and a DNA single-strand conformation polymorphism technique. The results are consistent with those obtained by Southern blotting in that sibship can be defined easily. A major advantage is that cells may be taken directly from the microtiter plate, eliminating the necessity to expand the clones and isolate genomic DNA. Cell lines which have not undergone receptor gene rearrangements cannot serve as PCR templates and do not interfere with this analysis. Furthermore, background from the large number of nonmutant lymphocytes present in the well does not hinder the analysis of the T cell receptor pattern of a mutant. This technique facilitates rapid screening of a large number of clones in a shorter time than Southern blotting, and is useful for the study of in vivo mutation and the clonal expansion of mutants in populations of T cells.
Caggana
M
M
Department of Cancer Biology, Harvard School of Public Health, Boston, MA 02115.
Benjamin
M B
MB
Little
J B
JB
Liber
H L
HL
Kelsey
K T
KT
D001483:Base Sequence; D015139:Blotting, Southern; D004277:DNA, Single-Stranded / Q000235:genetics*; D015329:Gene Rearrangement, T-Lymphocyte / Q000235:genetics*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004277:DNA, Single-Stranded; D007041:Hypoxanthine Phosphoribosyltransferase