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Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing
Published on: October 19, 2013
Multiple mutation analyses in single tumor cells with improved whole genome amplification
W Dietmaier1, A Hartmann, S Wallinger
1Institute of Pathology, the Department of Surgery, University of Regensburg, Germany. wolfgang.dietmaier@klinik.uni-regensburg.de
The American Journal of Pathology
|January 23, 1999
Summary
An improved whole genome amplification method, I-PEP-PCR, enhances DNA amplification from single cancer cells. This technique enables reliable mutation and microsatellite analysis, advancing molecular pathology diagnostics.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Whole genome amplification (WGA) combined with microdissection offers potential for cancer research.
- Existing WGA protocols require optimization for routine tumor pathology samples.
- Single-cell analysis is crucial for understanding cancer heterogeneity and progression.
Purpose of the Study:
- To evaluate established WGA protocols against an improved I-PEP-PCR method.
- To assess the feasibility of I-PEP-PCR for mutation analysis in tumor cells.
- To determine the efficiency and reliability of I-PEP-PCR for molecular analyses at the single-cell level.
Main Methods:
- Comparison of I-PEP-PCR with PEP-PCR and DOP-PCR using defined numbers of flow-sorted and microdissected tumor cells.
- Mutation analysis of p53 gene in breast carcinoma cells using I-PEP-PCR and conventional sequencing.
- Ki-ras mutation detection in single disseminated tumor cells from bone marrow aspirates using I-PEP-PCR and RFLP-PCR.
Main Results:
- I-PEP-PCR demonstrated superior DNA amplification efficiency from single and few cells compared to PEP-PCR and DOP-PCR.
- I-PEP-PCR achieved 100% sequence accuracy and enabled Ki-ras mutation detection in single cells.
- Reliable microsatellite analysis required 10-30 cells depending on sample type (fresh, frozen, or FFPE).
Conclusions:
- I-PEP-PCR is a powerful tool for molecular analyses in tumor pathology, including mutation and microsatellite instability detection.
- The method is effective even at the single-cell level, enabling detailed molecular profiling.
- I-PEP-PCR significantly advances the capabilities for diagnostic and experimental tumor pathology.
