High-resolution analysis of immunoglobulin heavy-chain gene rearrangements using denaturing gradient gel electrophoresis

A. Tierens, M. D. Lozano, R. Wickert, W. C. Chan, Timothy Charles Greiner

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

The detection of clonality in B-cell lymphomas has been facilitated by polymerase chain reaction (PCR) analysis of the immunoglobulin heavy-chain gene (IgH) complementarity determining region 3 (CDR3) and size fractionation by polyacrylamide gel electrophoresis (PAGE). However, the detection of minor clonal populations and biallelic rearrangements and the isolation of monoclonal products from gels are sometimes problematic. This study evaluated whether denaturing gradient gel electrophoresis (DGGE), a technique that separates DNA based on nucleotide sequence rather than length, could alleviate these problems. A total of 32 selected cases was studied with a diagnosis of monoclonal (n = 10), polyclonal (n = 9), and indeterminate (n = 13) IgH gene rearrangements, which were determined by analysis of seminested IgH CDR3 PCR products in 8% PAGE. These cases were evaluated using DGGE of seminested IgH CDR3 PCR products that included a 40- bp GC clamp on the Jh primer. DGGE allowed the discrimination of monoclonal populations in 9 of 13 cases where 8% PAGE results were indeterminate. In addition, DGGE demonstrated biallelic IgH rearrangements in three cases where 8% PAGE revealed only one predominant product. DGGE facilitated the purification and isolation of clonal IgH CDR3 products for sequencing without prior cloning. As an adaptation of current IgH PCR protocols, DGGE can enhance the construction of tumor-specific CDR3 primers/probes for investigations of minimal residual disease.

Original languageEnglish (US)
Pages (from-to)159-165
Number of pages7
JournalDiagnostic Molecular Pathology
Volume5
Issue number3
DOIs
StatePublished - Sep 1 1996

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Immunoglobulin Heavy Chain Genes
Denaturing Gradient Gel Electrophoresis
Gene Rearrangement
Complementarity Determining Regions
Polyacrylamide Gel Electrophoresis
Polymerase Chain Reaction
Residual Neoplasm
B-Cell Lymphoma
Population
Organism Cloning
Gels
DNA

Keywords

  • Denaturing gradient gel electrophoresis
  • Immunoglobulin gene rearrangements
  • Lymphoma
  • Polymerase chain reaction

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Biology
  • Cell Biology

Cite this

High-resolution analysis of immunoglobulin heavy-chain gene rearrangements using denaturing gradient gel electrophoresis. / Tierens, A.; Lozano, M. D.; Wickert, R.; Chan, W. C.; Greiner, Timothy Charles.

In: Diagnostic Molecular Pathology, Vol. 5, No. 3, 01.09.1996, p. 159-165.

Research output: Contribution to journalArticle

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