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

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Abstract

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 jinctional 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.

References (30)

  • S.G. Fischer et al.

    DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: Correspondence with melting theory

  • R. Fodde et al.

    Mutation Detection by denaturing gradient gel electrophoresis (DGGE)

    Hum. Mutat.

    (1994)
  • P. Labrune et al.

    Single-strand conformation polymorphism for detection of mutations and base substitutions in Phenylketonuria

    Am. J. Hum. Genet.

    (1991)
  • L.S. Lerman et al.

    Sequence-determined DNA separations

    Ann. Rev. Biophys. Bioeng.

    (1984)
  • S. Mashiyama et al.

    Detection of p53 gene mutations in human brain tumors by single strand conformation polymorphism analysis of polymerase chain reaction products

    Oncogene

    (1991)
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    Present address: Department of Immunology, Institute for Animal Science and Health-DLO, Lelystad, Netherlands.

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