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Abstract
Low yields of extracted cell-free DNA (cfDNA) from plasma limit continued development of liquid biopsy in cancer, especially in early-stage cancer diagnostics and cancer screening applications. We investigate a novel liquid-phase-based DNA isolation method that utilizes aqueous two-phase systems to purify and concentrate circulating cfDNA. The PHASIFY MAX and PHASIFY ENRICH kits were compared to a commonly employed solid-phase extraction method on their ability to extract cfDNA from a set of 91 frozen plasma samples from cancer patients. Droplet digital PCR (ddPCR) was used as the downstream diagnostic to detect mutant copies. Compared to the QIAamp Circulating Nucleic Acid (QCNA) kit, the PHASIFY MAX method demonstrated 60% increase in DNA yield and 171% increase in mutant copy recovery, and the PHASIFY ENRICH kit demonstrated a 35% decrease in DNA yield with a 153% increase in mutant copy recovery. A follow-up study with PHASIFY ENRICH resulted in the positive conversion of 9 out of 47 plasma samples previously determined negative with QCNA extraction (all with known positive tissue genotyping). Our results indicate that this novel extraction technique offers higher cfDNA recovery resulting in better sensitivity for detection of cfDNA mutations compared to a commonly used solid-phase extraction method.
Highlights
Isolation of cell-free DNA from blood or other body fluids from patients with cancer has revolutionized molecular cancer diagnostics and cancer care[1]
The PHASIFY method utilizes a series of aqueous two-phase systems (ATPSs) with unique, proprietary formulations to isolate, purify, and concentrate cell-free DNA (cfDNA) from a plasma sample
Other groups have utilized ATPSs comprised of surfactants, polymers, salts, and ionic liquids to isolate various biomarkers[11,12,13,14,15]
Summary
Isolation of cell-free DNA from blood or other body fluids from patients with cancer has revolutionized molecular cancer diagnostics and cancer care[1]. In patients with localized cancers, isolation yields are lower, often not exceeding 5 ng per mL of plasma, which limits its utility for applications such as detection of minimal residual disease, monitoring the efficacy of adjuvant therapy, or early cancer screening[7,8] This represents an unmet need for novel sample preparation methods that offer improved cfDNA and ctDNA recovery from plasma. The industry standard for nucleic acid sample preparation (including cfDNA) utilizes solid-phase extraction methods such as silica matrix or paramagnetic bead technology for purification These techniques have been proven to achieve high recovery and purity of total nucleic acids; they have significant limitations in capturing minute levels of DNA and low molecular weight DNA, which is a typical characteristic of cfDNA9. We extracted cfDNA from cancer patient plasma samples using the PHASIFY method and compared cfDNA yield, mutant copy yield, and overall mutation detection to that of solid-phase extraction
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