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, 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 V beta gene family sequences polymerase chain reaction (PCR) amplified TcR V beta 5.1/5.4 and V beta 14 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 V beta 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 V beta 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 detailed assessment of amplified TcR V beta 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.

Denaturing and non-denaturing gel electrophoresis as methods for the detection of junctional diversity in rearranged T cell receptor sequences.

Two nucleic acid gel electrophoresis techniques were tested as a possible tool for analyzing junctional diversity in rearranged T cell receptor (TcR) sequences in order to define the extent of T cell heterogeneity. For this purpose denaturing gradient gel electrophoresis (DGGE) as well as non-denaturing gel electrophoresis (nDGE) techniques have been studied. Detection of junctional diversity is based on mobility shifts, caused by nucleotide sequence polymorphism, of polymerase chain reaction amplified rearranged TcR sequences. DGGE as well as nDGE procedures are suitable for the detection of junctional diversity in TcR V gene family sequences based on sequence dependent separation. Compared to DGGE, nDGE of DNA is a relatively simple and rapid procedure, with a high separation potential. nDGE permits separation of double stranded (homoduplexes) and/or single stranded DNA molecules of the majority of TcR chain encoding sequences. Formation and detection of unique heteroduplex molecules combined with single stranded DNA molecule analysis in nDGE permits the recognition of the remaining sequences, thus providing additional information on the degree of T cell heterogeneity. In conclusion, these nucleic acid gel electrophoresis techniques allow a direct assessment of the heterogeneity and clonality of T cell populations by the detection of junctional diversity in TcR chain encoding sequences. This analysis can be performed without the need of cell propagation and/or cellular cloning procedures, thereby eliminating the risk of introducing technical artefacts.