title: Enhanced ratio of signals enables digital mutation scanning for rare allele detection.
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.
authors.affiliation: Division of DNA Repair and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts.
authors.forename: Elena
authors.initials: E
authors.lastname: Castellanos-Rizaldos
authors.affiliation: 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.
authors.forename: Cloud
authors.initials: C
authors.lastname: Paweletz
authors.affiliation: Division of DNA Repair and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts.
authors.forename: Chen
authors.initials: C
authors.lastname: Song
authors.affiliation: 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.
authors.forename: Geoffrey R
authors.initials: GR
authors.lastname: Oxnard
authors.affiliation: Department of Radiation Oncology, Dana-Farber Cancer Institute/Brigham and Women's Hospital Cancer Center, Harvard Medical School, Boston, Massachusetts.
authors.forename: Harvey
authors.initials: H
authors.lastname: Mamon
authors.affiliation: 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.
authors.forename: Pasi A
authors.initials: PA
authors.lastname: Jänne
authors.affiliation: 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.
authors.forename: G Mike
authors.initials: GM
authors.lastname: Makrigiorgos
mesh_terms: D000483:Alleles; D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D066246:ErbB Receptors / Q000235:genetics; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: D016159:Tumor Suppressor Protein p53; D004247:DNA; D066246:ErbB Receptors