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DMSO Increases Mutation Scanning Detection Sensitivity of High-Resolution Melting in Clinical Samples
Chen Song1, Elena Castellanos-Rizaldos1, Rafael Bejar2
1Department of Radiation Oncology, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, Boston, MA;
Clinical Chemistry
|October 4, 2015
Summary
Dimethyl sulfoxide (DMSO) enhances high-resolution melting (HRM) for mutation scanning, improving detection sensitivity. Combining DMSO with COLD-PCR significantly boosts the ability to detect low-abundance DNA variations, aiding clinical applications.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Mutation scanning using PCR-HRM is cost-effective for DNA variation identification.
- Conventional PCR-HRM has limitations in detecting low-abundance mutations (3%-10%).
- Enhancing HRM sensitivity is crucial for clinical applications.
Purpose of the Study:
- To improve the detection sensitivity of PCR-HRM for low-abundance mutations.
- To evaluate the impact of DMSO on HRM.
- To assess the utility of COLD-PCR combined with HRM for mutation enrichment.
Main Methods:
- Serial dilution of cell lines with TP53 exon 8 mutation.
- Application of DMSO in conventional PCR-HRM.
- Utilizing COLD-PCR and full-COLD-PCR for mutation enrichment.
- Employing droplet-digital PCR for optimization.
- Scanning TP53 exon 8 in cancer and myelodysplastic syndrome samples.
Main Results:
- DMSO increased PCR-HRM sensitivity 2- to 5-fold, detecting mutations down to approximately 1%.
- Full-COLD-PCR combined with DMSO-HRM detected mutations at 0.2%-0.3% abundance in TP53 exon 8.
- Overall sensitivity improvement of approximately 20-fold was achieved with full-COLD-PCR and DMSO-HRM.
Conclusions:
- DMSO significantly enhances HRM mutation scanning sensitivity.
- Full-COLD-PCR followed by DMSO-HRM offers a substantial improvement in detecting low-level mutations.
- These optimized methods have implications for clinical mutation detection.
