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• W2887762378 abstract "Presence of excess unaltered, wild-type DNA providing no information of biologic or clinical value may often mask rare alterations containing diagnostic or therapeutic clues for cancer. There is a growing demand for removing unaltered DNA over large pools-of-sequences, especially for applications in liquid biopsies with circulating DNA (cfDNA). We recently developed nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO), a single-step approach that removes WT-DNA prior to DNA amplification, following which current genomic analysis processes remain unchanged. NaME-PrO employs a double-strand DNA-specific nuclease (DSN) and overlapping oligonucleotide-probes interrogating multiple DNA targets. Following genomic-DNA denaturation, the temperature is lowered and the probes form transient double-stranded regions with their targets, and DSN is added to provide nuclease digestion to the selected sites. Mutations create mismatches that inhibit DSN digestion; thus, subsequent amplification yields DNA with alterations enhanced at multiple targets. In this manner, WT DNA at hundreds or thousands of DNA regions can be digested simultaneously. We also show that addition of organic solvents like DMSO in the NaME-PrO reaction enables DNA denaturation at lower temperatures that do not inactivate the enzyme (DSN). This enables DSN addition from the beginning of the reaction, enabling homogeneous, closed-tube application of the approach and avoids contamination-prone sample manipulations due to open-tube addition of DSN enzyme after denaturation. Using NaME-PrO ± DMSO we demonstrate several-hundred-fold multiplexed mutation enrichment in diverse human samples on multiple clinically relevant targets in 50-plex reactions. Application with targeted resequencing of tumor-circulating DNA demonstrated detection of mutations at the 0.01-0.1% levels with few sequence reads. In ongoing studies aiming to detect minimal residual disease using cfDNA in late-stage breast cancer, we used exome sequencing to identify multiple clonal mutations for individual patients. We then performed multiplexed NaME-PrO to trace the tumor fingerprint in plasma. In ~50-plex reactions we showed that the majority (>60%) of the tumor-specific mutations were also detected in plasma. To evaluate the ultimate sensitivity of the method to identify such tumor fingerprints, we applied NaME-PrO in serial dilutions of cfDNA from breast cancer patients into cfDNA from cancer-free, normal patients. We demonstrated that dilutions down to 1,000-10,000-fold, corresponding to mutations at Citation Format: Fangyan Yu, Mariana Fitarelli-Kiehl, Ka Wai Leong, Viktor Adalsteinsson, Heather Parsons, Mike G. Makrigiorgos. Method for sensitive detection of fingerprints in plasma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 941." @default.
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• W2887762378 date "2018-07-01" @default.
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• W2887762378 title "Abstract 941: Method for sensitive detection of tumor fingerprints in plasma" @default.
• W2887762378 doi "https://doi.org/10.1158/1538-7445.am2018-941" @default.
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