Direct assessment of junctional diversity in rearranged T cell receptor β chain encoding genes by combined heteroduplex and single strand conformation polymorphism (SSCP) analysis

Abstract

In order to define the extent of T cell heterogeneity and clonality, unique DNA sequences in the junctional region in rearranged T cell receptor (TcR) genes can be studied. For this purpose we have adapted a non-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 polymorphism. To examine the capacity of this nucleic acid gel electrophoresis procedure to detect nucleotide sequence polymorphism in the CDR 3 region within TcR Vβ gene family sequences polymerase chain reaction (PCR) amplified TcR Vβ5.1 5.4 and Vβ14 cDNA sequences were analyzed. The results of this study showed that (1) the single strand conformation polymorphism (SSCP) procedure has a low capacity to discriminate between diverse TcR Vβ cDNA sequences due to comigration 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 heteroduplex molecules; these heteroduplex molecules provide essential additional information on the degree of nucleotide sequence polymorphism in the CDR 3 region within the TcR Vβ cDNA sequences; (3) the double strand conformation polymorphism (DSCP) procedure provides a fast and reliable procedure to detect junctional diversity within the sequences tested. Using DSCP a more detained assessment of amplified TcR Vβ cDNA sequences can be obtained as compared with SSCP analysis only. Data obtained by gel analysis were very similar to those obtained by conventional bacterial cloning and DNA sequencing procedures on the corresponding cDNA clones. In conclusion, this new gel electrophoresis procedure allows a direct assessment of the extent of T cell heterogeneity and clonality by screening junctional diversity in TcR chain encoding sequences in clinical conditions with (oligo)clonal expansion of T lymphocytes.