Single-strand conformation polymorphisms can be used to detect T cell receptor gene rearrangements: an application to the in vivo hprt mutation assay

Abstract

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.

Southern blot analysis of T‐cell receptor gene rearrangements in cynomolgus monkeys, and identification of a progenitor cell HPRT mutation

“…Sufficient homology between human and monkey TCR segments allowed a gross comparison of monkey TCR banding patterns. We have chosen to analyze TCR patterns by Southern blotting in our work because genomic DNA sequence information is insufficient to allow use of PCR methods for TCR analysis [Bourguin et al, 1990;Caggana et al, 1991;Curry et al, 1993;de Boer et al, 19931. To our knowledge, this represents the first report of an analysis of monkey TCR restriction patterns.…”

Mutagenesis after cancer therapy.

“…We applied a technique using the polymerase chain reaction (amplifying a variable region within the T-cell receptor-y gene locus) combined with DNA single strand conformation polymorphism gel electrophoresis (PCR/SSCP) to determine the proportions of unique and sibling mutants that appeared in the cloning wells (42,43). We used this PCR/ SSCP technique to examine prospectively sets of mutants isolated from patients treated for HD and squamous cell carcinoma (SCC) of the head and neck after repeated samplings.…”

Multiplex polymerase chain reaction-based analysis of T-cell receptor ? gene rearrangements for the determination of T-lymphocyte clonality

“…This may lead to an overestimation of mutant frequencies and an altered mutational spectrum caused by clonally expanded "hotspot" mutants [Nicklas et al, 1988;Hakoda et al, 1989;O'Neill et al, 1994]. Because the structure and DNA sequence of the T-cell receptor (TCR) genes is different in each T-lymphocyte, analysis of rear-rangements of the TCR genes has been used for determination of the clonality of hprt mutant T-lymphocytes [Clark and Nicklas, 1996;Nicklas et al, 1986;Caggana et al, 1991;de Boer et al, 1993] and mutant lymphocytes at other loci, such as the HLA-A gene [Grist et al, 1992]. (Note that the TCR ␥ gene consists of variable (V), joining (J), and constant (C) gene segments.…”

“…Although the TCR ␥ gene is suitable for PCR-based analysis due to its small number of subgroups, the total number of PCR reactions required to include all possible types of recombinations is still very large. The PCR-based method for determination of hprt mutant T-lymphocyte clonality uses primers only for V␥I and J␥1/2 subgroups of the TCR ␥ gene [Caggana et al, 1991;de Boer et al, 1993]. Clones with rearranged TCR ␥ genes involving other subgroups of V and J gene segments cannot be analyzed.…”

“…Clones with rearranged TCR ␥ genes involving other subgroups of V and J gene segments cannot be analyzed. In addition, further analysis of the PCR-amplified TCR ␥ gene products is required to determine the clonality of mutant lymphocytes using singlestrand conformation polymorphism (SSCP), restriction fragment length polymorphism (RFLP), or direct sequence analysis [Clark and Nicklas, 1996;Caggana et al, 1991;Curry et al, 1993;de Boer et al, 1993].…”