DIRECT ASSESSMENT OF JUNCTIONAL DIVERSITY IN REARRANGED T-CELL RECEPTOR-BETA CHAIN ENCODING GENES BY COMBINED HETERODUPLEX AND SINGLE-STRAND CONFORMATION POLYMORPHISM (SSCP) ANALYSIS

In order to define the extent of T cell heterogeneity and clonality, u nique DNA sequences in the junctional region in rearranged T cell rece ptor (TcR) genes can be studied. For this purpose we have adapted a no n-denaturing nucleic acid gel electrophoresis procedure to detect TcR junctional diversity. Detection of junctional diversity is based upon electrophoretic separation of single stranded (ss) and double stranded (ds) DNA molecules via mobility shifts due to nucleotide sequence pol ymorphism. To examine the capacity of this nucleic acid gel electropho resis procedure to detect nucleotide sequence polymorphism in the CDR 3 region within TcR V beta gene family sequences polymerase chain reac tion (PCR) amplified TcR V beta 5.1/5.4 and V beta 14 cDNA sequences w ere analyzed, The results of this study showed that (1) the single str and conformation polymorphism (SSCP) procedure has a low capacity to d iscriminate between diverse TcR V beta cDNA sequences due to comigrati on of the ssDNA molecules, which results in an underestimation of the heterogeneity in a given T cell population; (2) comigrating ssDNA and/ or dsDNA (homoduplex) molecules can be separated by the formation of h eteroduplex molecules; these heteroduplex molecules provide essential additional information on the degree of nucleotide sequence polymorphi sm in the CDR 3 region within the TcR V beta cDNA sequences; (3) the d ouble strand conformation polymorphism (DSCP) procedure provides a fas t and reliable procedure to detect junctional diversity within the seq uences tested. Using DSCP a more detailed assessment of amplified TcR V beta cDNA sequences can be obtained as compared with SSCP analysis o nly, Data obtained by gel analysis were very similar to those obtained by conventional bacterial cloning and DNA sequencing procedures on th e corresponding cDNA clones. In conclusion, this new gel electrophores is procedure allows a direct assessment of the extent of T cell hetero geneity and clonality by screening junctional diversity in TcR chain e ncoding sequences in clinical conditions with (oligo)clonal expansion of T lymphocytes.