An improved PCR-heteroduplex method permits high-sensitivity detection of clonal expansions in complex T cell populations

J Immunol Methods. 1996 Sep 27;196(2):181-92. doi: 10.1016/0022-1759(96)00114-7.


We have modified the PCR-heteroduplex technique to render it more suitable for the study of the clonal make up of complex T cell populations. This technique is based on separate PCR amplifications of all the TCR V beta genes expressed by a polyclonal T cell sample, followed by a heteroduplex reaction of the PCR products and a gel separation. Our modification involves performing each heteroduplex reaction in the presence of excess carrier DNA, which is the PCR product of a cloned TCR V beta cDNA having the same variable and constant region of the amplified V beta family, but a different N region. In this way, every clonotypic V beta chain that is amplified in the polyclonal mixture forms a unique and reproducible pair of heteroduplex bands with the carrier DNA. This molecular footprint permits the identification of a given T cell clone over time, or in different anatomical sites. The specificity and sensitivity of the detection of T cell clones can be further increased by hybridising the blotted heteroduplex gel with oligonucleotides specific for either a TCR V beta N region or the carrier DNA. In conclusion, we have developed a simple and reproducible technique that permits the simultaneous detection of the expanded T cell clones present in heterogeneous T cell populations in a very specific and sensitive manner.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aged
  • Base Sequence
  • CD4-Positive T-Lymphocytes / immunology
  • Carrier Proteins / genetics
  • Clone Cells
  • DNA / chemistry
  • Humans
  • Influenza Vaccines / immunology
  • Leukocyte Common Antigens / analysis
  • Leukocyte Common Antigens / genetics
  • Lymphocyte Activation*
  • Molecular Sequence Data
  • Nucleic Acid Heteroduplexes / analysis*
  • Polymerase Chain Reaction / methods*
  • Receptors, Antigen, T-Cell / genetics
  • Sensitivity and Specificity
  • T-Lymphocyte Subsets / cytology
  • T-Lymphocyte Subsets / immunology*


  • Carrier Proteins
  • Influenza Vaccines
  • Nucleic Acid Heteroduplexes
  • Receptors, Antigen, T-Cell
  • DNA
  • Leukocyte Common Antigens