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Polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE): sensitivity, band pattern analysis, and
1Department of Dermatology, Skin Diseases Research Center at Case Western Reserve University, Cleveland, Ohio, USA.
The American Journal of Dermatopathology
|December 23, 1999
Summary
Polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE) can detect T-cell clonality with a 0.1%-1% threshold. Optimization minimizes artifactual bands, confirming multiple bands can arise from a single T-cell receptor gamma gene rearrangement.
Area of Science:
- Molecular Biology
- Immunology
- Genetics
Background:
- Polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE) is increasingly used for T-cell clonality assessment in challenging samples like skin biopsies.
- T-cell receptor (TCR)-gamma gene rearrangements are analyzed via nested PCR and DGGE, offering improved sensitivity over Southern blot but with undefined limitations.
- The presence of secondary bands alongside primary bands in PCR/DGGE analysis complicates interpretation of T-cell clonality.
Purpose of the Study:
- To define the clonal detection threshold of PCR/DGGE for T-cell populations.
- To elucidate the origin of multiple bands observed in TCR-gamma gene rearrangement analysis.
- To optimize PCR/DGGE methods for reducing artifactual bands and improving accuracy.
Main Methods:
- Nested PCR amplification of TCR-gamma gene rearrangements using consensus primers, followed by separation using DGGE.
- Titration studies using varying ratios of monoclonal T-cell DNA and polyclonal background DNA (keratinocyte and tonsil DNA).
- Analysis of monoclonal T-cell lines and manipulation of PCR conditions (e.g., template amount, primer concentration) to investigate band formation.
Main Results:
- The sensitivity of PCR/DGGE is influenced by the polyclonal T-cell background, with detection thresholds ranging from 0.001% (keratinocyte DNA) to 1% (tonsil DNA).
- A single TCR-gamma gene rearrangement can generate multiple bands, identified as an artifact of the nested PCR process.
- Reducing the amount of first-round PCR product or decreasing first-round primer concentration significantly minimized secondary bands without affecting primary band intensity.
Conclusions:
- PCR/DGGE offers a realistic clonal detection threshold of 0.1% to 1% for T-cell populations.
- Multiple bands in PCR/DGGE are not indicative of multiple clones but can be consistent with a monoclonal T-cell population.
- Optimized PCR/DGGE conditions can effectively minimize artifactual secondary bands, enhancing the reliability of T-cell clonality assessment.