Detection and characterization of alpha-beta-T-cell clonality by denaturing gradient gel electrophoresis (DGGE)

Biotechniques. 1998 Aug;25(2):244-50. doi: 10.2144/98252st05.

Abstract

Accumulation of T cells carrying identical T-cell receptors (TCR) is associated with a number of immunological and non-immunological diseases. Therefore, it is of interest to be able to analyze complex T-cell populations for the presence of clonally expanded subpopulations. Here, we describe a simple method combining reverse transcription (RT)-PCR and denaturing gradient gel electrophoresis (DGGE) for rapid detection and characterization of T-cell clonality. The detection of clonally expanded T cells by DGGE relies on the fact that clonal transcripts have no junctional diversity and therefore resolve at a fixed position in the gel, which is determined by their melting properties. For polyclonal populations with a high degree of junctional diversity, the different DNA molecules will resolve at different positions in the gel and together will be revealed as a smear. For each of the TCR beta-variable gene (BV) 1-24 families, cloned transcripts were amplified and shown to resolve as distinct bands in the denaturing gradient gel, whereas the analysis of polyclonal T-cell populations resulted in a smear in the gel. The present method might prove useful to test for clonotypic T-cells in a variety of pathological and physiological conditions and for monitoring T-cell responses in diagnostic and therapeutic settings.

Publication types

  • Research Support, Non-U.S. Gov't
  • Technical Report

MeSH terms

  • Cells, Cultured
  • Clone Cells
  • DNA Primers
  • Electrophoresis, Polyacrylamide Gel / methods*
  • Humans
  • Nucleic Acid Denaturation*
  • Receptors, Antigen, T-Cell, alpha-beta / chemistry
  • Receptors, Antigen, T-Cell, alpha-beta / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction
  • T-Lymphocytes / metabolism*
  • Transcription, Genetic

Substances

  • DNA Primers
  • Receptors, Antigen, T-Cell, alpha-beta