Proof of concept for aqueous two-phase system-based extraction of cell-free DNA from plasma for liquid biopsy...

Efficient extraction of circulating cell-free DNA (cfDNA) from plasma is critical for liquid biopsy applications, but remains challenging due to cfDNA's short fragment size, low concentration, and the presence of interfering proteins and genomic DNA. This study presents a simple extraction process based on aqueous two-phase system (ATPS) capture and reverse elution purification as a potentially lysis-free alternative to adsorption-based methods. Short DNA fragments selectively partitioned into the salt-rich bottom phase, achieving up to 65% recovery after purification while removing 99.7% of plasma proteins. Reverse elution enabled efficient desalting and up to fourfold DNA concentration under mild, aqueous conditions. The extracts showed no qPCR inhibition and displayed a cfDNA-like size profile with a cutoff around 750 bp, indicating selective enrichment of short fragments. Although the total yield was slightly lower than that of the QIAamp Circulating Nucleic Acid Kit (QIAGEN), the ATPS workflow substantially reduced processing time, equipment needs, and cost. Next-generation sequencing of cfDNA reference material extracted from DNA-free plasma confirmed compatibility with library preparation and sequencing workflows, providing proof of concept for a simplified cfDNA extraction strategy for amplification-based liquid biopsy applications.