Send to

Choose Destination
Clin Chem. 2015 Nov;61(11):1354-62. doi: 10.1373/clinchem.2015.245357. Epub 2015 Oct 2.

DMSO Increases Mutation Scanning Detection Sensitivity of High-Resolution Melting in Clinical Samples.

Author information

Department of Radiation Oncology, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, Boston, MA;
Division of Hematology and Oncology, UCSD Moores Cancer Center, La Jolla, CA;
Division of Hematology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.
Department of Radiation Oncology, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, Boston, MA;



Mutation scanning provides the simplest, lowest-cost method for identifying DNA variations on single PCR amplicons, and it may be performed before sequencing to avoid screening of noninformative wild-type samples. High-resolution melting (HRM) is the most commonly used method for mutation scanning. With PCR-HRM, however, mutations less abundant than approximately 3%-10% that can still be clinically significant may often be missed. Therefore, enhancing HRM detection sensitivity is important for mutation scanning and its clinical application.


We used serial dilution of cell lines containing the TP53 exon 8 mutation to demonstrate the improvement in detection sensitivity for conventional-PCR-HRM in the presence of DMSO. We also conducted coamplification at lower denaturation temperature (COLD)-PCR with an extra step for cross-hybridization, followed by preferential denaturation and amplification at optimized critical temperature (full-COLD-PCR), to further enrich low-level mutations before HRM with or without DMSO, and we used droplet-digital PCR to derive the optimal conditions for mutation enrichment. Both conventional PCR-HRM and full-COLD-PCR-HRM with and without DMSO were used for mutation scanning of TP53 exon 8 in cancer samples containing known mutations and myelodysplastic syndrome samples with unknown mutations. Mutations in other genes were also examined.


The detection sensitivity of PCR-HRM scanning increases 2- to 5-fold in the presence of DMSO, depending on mutation type and sequence context, and can typically detect mutation abundance of approximately 1%. When mutation enrichment is applied during amplification with full-COLD-PCR followed by HRM in the presence of DMSO, mutations with 0.2%-0.3% abundance in TP53 exon 8 can be detected.


DMSO improves HRM mutation scanning sensitivity with saturating dyes. When full-COLD-PCR is used, followed by DMSO-HRM, the overall improvement is about 20-fold compared with conventional PCR-HRM.

[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center