Selective capture of plasma cell-free tumor DNA on magnetic beads: a sensitive and versatile tool for liquid biopsy
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Selective capture of plasma cell-free tumor DNA on magnetic beads: a sensitive and versatile tool for liquid biopsy Mohammad Amin Kerachian 1,2,3 & Marjan Azghandi 3,4 & Ali Javadmanesh 3,4 & Kamran Ghaffarzadegan 5,6 & Sina Mozaffari-Jovin 1,2,7 Received: 30 November 2019 / Revised: 6 May 2020 / Accepted: 15 May 2020 # International Society for Cellular Oncology 2020
Abstract Purpose Recently, ‘solid tumor biopsies’ have been challenged by the emergence of ‘liquid biopsies’, which are aimed at the isolation and detection of circulating cell-free tumor DNA (ctDNA) in body fluids. Here, we developed and optimized a method for selective capture of ctDNA on magnetic beads (SCC-MAG) for mutation detection in plasma of patients with colorectal cancer (CRC). Methods Blood and tissue samples from 28 CRC patients were included for the detection of KRAS mutations. For the tissue samples, mutation analysis was conducted by high resolution melting (HRM) analysis and sequencing. For the SCCMAG method, ctDNA was isolated from 200 µl plasma from patients with a mutant KRAS gene. For comparison, ctDNA extraction was carried out using a silica membrane-based method, after which mutations were detected using Intplex allele-specific PCR. Results The mean ctDNA integrity index in plasma samples of cancer patients was 1.03, comparable with that of silica membrane-derived ctDNA (1.011). Notably, the limit of detection for the SCC-MAG approach was lower than that of the silica membrane method and measured 2.25 pg/ml ctDNA in plasma. Our analyses showed that while the silica membrane-based approach was capable of collecting ctDNA from two out of six CRC patient samples (average Cq 34.23), the SCC-MAG captured ctDNA from all samples with an average Cq of 29.76. Conclusions We present a robust, reproducible, and highly sensitive method for the analysis of mutation statuses in liquid biopsies. The SCC-MAG method can readily be applied to any nucleic acid target for diagnostic purposes upon careful design of the specific capture probes, and can be multiplexed by several probes to identify multiple targets. Keywords Cell-free tumor DNA extraction . ctDNA . Colorectal cancer . KRAS mutations . Plasma . Serum
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13402-020-00536-2) contains supplementary material, which is available to authorized users. * Mohammad Amin Kerachian [email protected]; [email protected]
4
Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
5
Department of Pathology and Laboratory Medicine, Mayo Clinic, Arizona, Scottsdale, AZ, USA
Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
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Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Azadi Square, Mashhad 917794-8564, Iran
Razavi Cancer Research Center, Razavi Hospital, Imam Reza International University, Mashhad, Iran
7
Department of Cellular Biochemist
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