High-resolution analysis of immunoglobulin heavy-chain gene rearrangements using denaturing gradient gel electrophoresis.
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.
Tierens
A
A
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha 68198-3135, USA.
Lozano
M D
MD
Wickert
R
R
Chan
W C
WC
Greiner
T C
TC
D000911:Antibodies, Monoclonal / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D015322:Gene Rearrangement, B-Lymphocyte / Q000235:genetics*; D005803:Genes, Immunoglobulin; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains