Format

Send to

Choose Destination
J Mol Diagn. 2015 May;17(3):284-92. doi: 10.1016/j.jmoldx.2014.12.003. Epub 2015 Mar 13.

Enhanced ratio of signals enables digital mutation scanning for rare allele detection.

Author information

1
Division of DNA Repair and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts.
2
Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts; Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts.
3
Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts; Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts.
4
Department of Radiation Oncology, Dana-Farber Cancer Institute/Brigham and Women's Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts.
5
Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts; Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts; Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts.
6
Division of DNA Repair and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts; Department of Radiation Oncology, Dana-Farber Cancer Institute/Brigham and Women's Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts. Electronic address: mmakrigiorgos@lroc.harvard.edu.

Abstract

The use of droplet digital PCR (ddPCR) for low-level DNA mutation detection in cancer, prenatal diagnosis, and infectious diseases is growing rapidly. However, although ddPCR has been implemented successfully for detection of rare mutations at pre-determined positions, no ddPCR adaptation for mutation scanning exists. Yet, frequently, clinically relevant mutations reside on multiple sequence positions in tumor suppressor genes or complex hotspot mutations in oncogenes. Here, we describe a combination of coamplification at lower denaturation temperature PCR (COLD-PCR) with ddPCR that enables digital mutation scanning within approximately 50-bp sections of a target amplicon. Two FAM/HEX-labeled hydrolysis probes matching the wild-type sequence are used during ddPCR. The ratio of FAM/HEX-positive droplets is constant when wild-type amplicons are amplified but deviates when mutations anywhere under the FAM or HEX probes are present. To enhance the change in FAM/HEX ratio, we employed COLD-PCR cycling conditions that enrich mutation-containing amplicons anywhere on the sequence. We validated COLD-ddPCR on multiple mutations in TP53 and in EGFR using serial mutation dilutions and cell-free circulating DNA samples, and demonstrate detection down to approximately 0.2% to 1.2% mutation abundance. COLD-ddPCR enables a simple, rapid, and robust two-fluorophore detection method for the identification of multiple mutations during ddPCR and potentially can identify unknown DNA variants present in the target sequence.

PMID:
25772705
PMCID:
PMC4411249
DOI:
10.1016/j.jmoldx.2014.12.003
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center