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 language | English (US) |
|---|---|
| Pages (from-to) | 159-165 |
| Number of pages | 7 |
| Journal | Diagnostic Molecular Pathology |
| Volume | 5 |
| Issue number | 3 |
| DOIs | |
| State | Published - Sep 1 1996 |
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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 journal › Article
}
TY - JOUR
T1 - High-resolution analysis of immunoglobulin heavy-chain gene rearrangements using denaturing gradient gel electrophoresis
AU - Tierens, A.
AU - Lozano, M. D.
AU - Wickert, R.
AU - Chan, W. C.
AU - Greiner, Timothy Charles
PY - 1996/9/1
Y1 - 1996/9/1
N2 - 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.
AB - 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.
KW - Denaturing gradient gel electrophoresis
KW - Immunoglobulin gene rearrangements
KW - Lymphoma
KW - Polymerase chain reaction
UR - http://www.scopus.com/inward/record.url?scp=0029811894&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0029811894&partnerID=8YFLogxK
U2 - 10.1097/00019606-199609000-00003
DO - 10.1097/00019606-199609000-00003
M3 - Article
C2 - 8866228
AN - SCOPUS:0029811894
VL - 5
SP - 159
EP - 165
JO - Diagnostic Molecular Pathology
JF - Diagnostic Molecular Pathology
SN - 1052-9551
IS - 3
ER -