DENATURING AND NONDENATURING 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 possi ble tool for analyzing junctional diversity in rearranged T cell recep tor (TcR) sequences in order to define the extent of T cell heterogene ity. For this purpose denaturing gradient gel electrophoresis (DGGE) a s well as non-denaturing gel electrophoresis (nDGE) techniques have be en studied. Detection of junctional diversity is based on mobility shi fts, caused by nucleotide sequence polymorphism, of polymerase chain r eaction amplified rearranged TcR sequences. DGGE as well as nDGE proce dures 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 strand ed (homoduplexes) and/or single stranded DNA molecules of the majority of TcR chain encoding sequences. Formation and detection of unique he teroduplex molecules combined with single stranded DNA molecule analys is in nDGE permits the recognition of the remaining sequences, thus pr oviding 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 popu lations by the detection of junctional diversity in TcR chain encoding sequences. This analysis can be performed without the need of cell pr opagation and/or cellular cloning procedures, thereby eliminating the risk of introducing technical artefacts.