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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

1
Department of Radiation Oncology, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, Boston, MA;
2
Division of Hematology and Oncology, UCSD Moores Cancer Center, La Jolla, CA;
3
Division of Hematology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.
4
Department of Radiation Oncology, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, Boston, MA; mmakrigiorgos@lroc.harvard.edu.

Abstract

BACKGROUND:

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.

METHODS:

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.

RESULTS:

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.

CONCLUSIONS:

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.

PMID:
26432802
PMCID:
PMC5156568
DOI:
10.1373/clinchem.2015.245357
[Indexed for MEDLINE]
Free PMC Article

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