Rapid Electrokinetic Isolation of Cancer-Related Circulating Cell-Free DNA Directly from Blood

Abstract

BACKGROUND Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a “liquid biopsy” may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. METHODS We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification,PCR,and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced. RESULTS PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15–20 mL blood. CONCLUSIONS Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring.

Circulating Tumor DNA as a Liquid Biopsy for Cancer

“…Selection of an isolation method that ensures extraction of a sufficient amount of high-quality DNA is critical and it has been shown that preanalytical factors of blood sampling and processing can strongly affect DNA yield (36 ). Because many conventional methods for the isolation of cfDNA are costly, timeconsuming, and complex, Sonnenberg et al developed a novel electrokinetic technique that allowed rapid isolation of cfDNA directly from blood (37 ). Second, one of the most important issues is the lack of harmonization of quantification methods.…”

Circulating Tumor Cells Versus Circulating Tumor DNA in Colorectal Cancer: Pros and Cons

“…However, more recently, this process has been simplified with the availability of various commercial kits, such as QIAmp 96 spin blood DNA extraction kit, QIAmp DNA blood mini kit, Bilatest DNA kit, Quanti-iT™ DNA high-sensitivity assay kit. Sonnenberg et al recently described a novel electrokinetic technique with an AC electrokinetic microarray device (Biological Dynamics) that allows for rapid isolation of circulating DNA from blood [67]. Unfortunately, these isolation and detection procedures are not standardized, and none of these technologies are FDA approved.…”

Dielectrophoresis for Biomedical Sciences Applications: A Review

“…Furthermore, unlike optical tweezers, DEP provides a low applied voltage and an alternating current for gene transfection from DNA that can prevent cell membrane damage [165]. DEP techniques use a smaller sample volume and can isolate to the extent of a nanoscale-sized bio-particle, making it an excellent candidate for further development as a point-of-care (POC) diagnostic tool, especially in molecular research [176,177,178]. …”