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PLoS One. 2015 Nov 12;10(11):e0142273. doi: 10.1371/journal.pone.0142273. eCollection 2015.

SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients.

Author information

1
Biotechnology in Animal Production, Department for Agrobiotechnology, IFA Tulln, Tulln, Lower Austria, Austria.
2
Institute of Animal Breeding and Genetics, University of Veterinary Medicine Vienna, Vienna, Austria.
3
Department of Dermatology and Venereology, Karl Landsteiner University of Health Sciences, St. Poelten, Lower Austria, Austria.
4
Karl Landsteiner Institute of Dermatological Research, St. Poelten, Lower Austria, Austria.
5
Research Group Oncology of the Equine Clinic, Department for Companion Animal and Horses, University of Veterinary Medicine Vienna, Vienna, Austria.
6
Department of Dermatology and Venereology, Landesklinikum Wiener Neustadt, Wiener Neustadt, Lower Austria, Austria.

Abstract

Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent "gold standard". Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution), at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients.

PMID:
26562020
PMCID:
PMC4642939
DOI:
10.1371/journal.pone.0142273
[Indexed for MEDLINE]
Free PMC Article

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