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Publication type: Journal Article
Document type: Full Paper

Year: 2015

Author(s): Stadler, J; Eder, J; Pratscher, B; Brandt, S; Schneller, D; Müllegger, R; Vogl, C; Trautinger, F; Brem, G; Burgstaller, JP

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

Source: PLoS One. 2015; 10(11):e0142273



Authors Vetmeduni Vienna:

Brandt Sabine
Brem Gottfried
Burgstaller Jörg
Pratscher Barbara
Schneller Doris
Stadler Julia Anna
Vogl Claus

Vetmed Research Units
University Equine Clinic, Clinical Unit of Equine Surgery
Institute of Animal Breeding and Genetics, Unit of Molecular Genetics
Institute of Animal Breeding and Genetics, Unit of Reproductive Biology


Project(s): DNA mutations in cancer: A personalised approach to assess tumour dynamics in melanoma


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.


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