ArticleThe Journal of molecular diagnostics : JMD2015
Enhanced ratio of signals enables digital mutation scanning for rare allele detection.
Article in The Journal of molecular diagnostics : JMD, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
12 citing papers in PubMed, 28 citations in OpenAlex.
- Combining E-ice-COLD-PCR and Pyrosequencing with Di-Base Addition (PDBA) Enables Sensitive Detection of Low-Abundance Mutations.Applied biochemistry and biotechnology · 2024Article
- Clinical impact of circulating tumor DNA to track minimal residual disease in colorectal cancer patients. Hopes and limitations.ESMO gastrointestinal oncology · 2024Review
- Triple Negative Breast Cancer: A Review of Present and Future Diagnostic Modalities.Medicina (Kaunas, Lithuania) · 2021Review
- Digital PCR as an Emerging Tool for Monitoring of Microbial Biodegradation.Molecules (Basel, Switzerland) · 2020Review
- Denaturation-Enhanced Droplet Digital PCR for Liquid Biopsies.Clinical chemistry · 2018Article
- Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics.Cancer medicine · 2017Article
- The feasibility of using mutation detection in ctDNA to assess tumor dynamics.International journal of cancer · 2017Review
- COLD-PCR Technologies in the Area of Personalized Medicine: Methodology and Applications.Molecular diagnosis & therapy · 2017Review
- The potential for liquid biopsies in the precision medical treatment of breast cancer.Cancer biology & medicine · 2016Article
- Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR.The Journal of molecular diagnostics : JMD · 2016Article
- DMSO Increases Mutation Scanning Detection Sensitivity of High-Resolution Melting in Clinical Samples.Clinical chemistry · 2015Article
- Circulating Cell-Free Tumour DNA in the Management of Cancer.International journal of molecular sciences · 2015Review
Corrections and comments
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Authors and funding
7 authors at 4 institutions in 1 country.
Funding
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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.