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Rapid and Efficient Extraction of Cell-Free DNA Using Homobifunctional Crosslinkers.

ABSTRACT: Since its discovery in circulating blood seven decades ago, cell-free DNA (cfDNA) has become a highly focused subject in cancer management using liquid biopsy. Despite its clinical utility, the extraction of cfDNA from blood has many technical difficulties, including a low efficiency of recovery and long processing times. We introduced a magnetic bead-based cfDNA extraction method using homobifunctional crosslinkers, including dimethyl suberimidate dihydrochloride (DMS). Owing to its bifunctional nature, DMS can bind to DNA through either covalent or electrostatic bonding. By adopting amine-conjugated magnetic beads, DMS-DNA complexes can be rapidly isolated from blood plasma. Using standard washing and eluting processes, we successfully extracted cfDNA from plasma within 10 min. This method yielded a 56% higher extraction efficiency than that of a commercial product (QIAamp kit). Furthermore, the instant binding mechanism of amine coupling between the microbeads and DMS-DNA complexes significantly reduced the processing time. These results highlight the potential of this magnetic bead-based homobifunctional crosslinker platform for extraction of cfDNA from blood plasma.

SUBMITTER: Seong H

PROVIDER: S-EPMC9405790 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Rapid and Efficient Extraction of Cell-Free DNA Using Homobifunctional Crosslinkers.

Seong HyeonAh H Park Junsoo J Bae Minju M Shin Sehyun S

Biomedicines 20220804 8

Since its discovery in circulating blood seven decades ago, cell-free DNA (cfDNA) has become a highly focused subject in cancer management using liquid biopsy. Despite its clinical utility, the extraction of cfDNA from blood has many technical difficulties, including a low efficiency of recovery and long processing times. We introduced a magnetic bead-based cfDNA extraction method using homobifunctional crosslinkers, including dimethyl suberimidate dihydrochloride (DMS). Owing to its bifunctiona ...[more]

PMID: 36009429

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Project description:Cell-free DNA (cfDNA) is a liquid biopsy marker that can carry signatures (i.e., mutations) associated with certain pathological conditions. Therefore, the extraction of cfDNA from a variety of clinical samples can be an effective and minimally invasive source of markers for disease detection and subsequent management. In the oncological diseases, circulating tumor DNA (ctDNA), a cfDNA sub-class, can carry clinically actionable mutations and coupled with next generation sequencing or other mutation detection methods provide a venue for effective in vitro diagnostics. However, cfDNA mutational analyses require high quality inputs. This necessitates extraction platforms that provide high recovery over the entire ctDNA size range (50 ? 150 bp) with minimal interferences (i.e., co-extraction of genomic DNA), and high reproducibility with a simple workflow. Herein, we present a novel microfluidic solid-phase extraction device (?SPE) consisting of a plastic chip that is activated with UV/O3 to generate surface-confined carboxylic acid functionalities for the ?SPE of cfDNA. The ?SPE uses an immobilization buffer (IB) consisting of polyethylene glycol and salts that induce cfDNA condensation onto the activated plastic microfluidic surface. The ?SPE consists of an array of micropillars to increase extraction bed load (scalable to loads >700 ng of cfDNA) and can be produced at low-cost using replication-based techniques. The entire ?SPE can be fabricated in a single molding step negating the need for adding additional extraction supports to the device simplifying production and keeping device and assay cost low. The ?SPE allowed for recoveries >90% of model cfDNA fragments across a range of sizes (100-700 bp) and even the ability to extract efficiently short cfDNA fragments (50 bp, >70%). In addition, the composition of the IB allowed for reducing the interference of co-extracted genomic DNA. We demonstrated the clinical utility of the ?SPE by quantifying the levels of cfDNA in healthy donors and patients with non-small-cell lung and colorectal cancers. ?SPE extracted cfDNA from plasma samples was also subjected to a ligase detection reaction (LDR) for determining the presence of mutations in the KRAS gene for colorectal and non-small cell lung cancer patients.

Project description:BackgroundFebrile haematological patients are at high risk for potential bloodstream infections, the rapid and accurate identification of pathogens is crucial for clinical diagnosis and treatment. Droplet Digital PCR (ddPCR) is a novel and ultra-sensitively molecular technique for the rapid detection of pathogens. We evaluated the ability of ddPCR to identify infectious etiologies to discuss the applicability of ddPCR in the diagnosis and treatment of infections for febrile haematological patients.MethodsThis study enrolled and analyzed 89 ddPCR tests performed on 71 febrile haematological patients. We conducted a comparison between ddPCR results, blood culture (BC), and conventional microbiological testing (CMT). Additionally, we analyzed the correlation between ddPCR results and inflammatory factors, as well as their impact on antimicrobial therapy.ResultsDdPCR detected 113 pathogens in 72 plasma samples, while CMT identified 39 pathogens in 32 plasma samples. The detection rate of bacteria and viruses using ddPCR was significantly higher than that of CMT (p <0.0001). The turnaround time (TAT) for pathogenic diagnosis was significantly shorter with ddPCR compared to CMT (p <0.0001). When we used the CMT as reference standard, the sensitivity and specificity of ddPCR were 93.8%, 26.3%, respectively. We observed a positive correlation between the ddPCR results and CRP, PCT and IL-6, and ddPCR (AUC=0.771) has better diagnostic performance. The anti-infective treatment strategies were adjusted for 30 patients based on the positive ddPCR results, with 86.7% (26/30) of these cases demonstrating effectiveness in the anti-infective treatment.ConclusionDdPCR has the potential to enhance pathogen detection in febrile haematological patients by offering high sensitivity, rapid, precise results, it demonstrates better diagnostic performance compared to inflammatory factors and can contribute to the real-time clinical optimization of antimicrobial regimens, thereby enhancing the efficacy of anti-infective therapy.

Dataset Information

Rapid and Efficient Extraction of Cell-Free DNA Using Homobifunctional Crosslinkers.

Publications

Rapid and Efficient Extraction of Cell-Free DNA Using Homobifunctional Crosslinkers.

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