Centrifugation-free extraction of circulating nucleic acids using immiscible liquid under vacuum pressure

Hoyoon Lee,Kyong Hwa Park,Wonhwi Na,Chanhee Park,Sehyun Shin

doi:10.1038/s41598-018-23766-9

Abstract

Extraction of cell-free DNA (cfDNA), which exists at an extremely low concentration in plasma, is a critical process for either targeted-sensing or massive sequencing of DNAs. However, such small amount of DNA cannot be fully obtained without high-speed centrifugation (<20,000 g). Here, we developed a centrifugation-free cfDNA extraction method and system that utilizes an immiscible solvent under single low vacuum pressure throughout the entire process. It has been named Pressure and Immiscibility-Based EXtraction (PIBEX). The amounts of extracted cfDNA by PIBEX were compared with those extracted by the conventional gold standards such as QIAGEN using quantitative PCR (qPCR). The PIBEX system showed equal performance regarding extraction amount and efficiency compared to the existing method. Because the PIBEX eliminates the troublous and repetitive centrifugation processes in DNA extraction, it can be further utilized in microfluidic-sample preparation systems for circulating nucleic acids, which would lead to an integrated sample-to-answer system in liquid biopsies.

Highlights

Nucleic acid-based molecular diagnosis is a key technology for implementing precision medicine in clinics[1,2,3]
Cell-free DNA, which freely circulates in the blood stream, has been highlighted as a potential biomarker of liquid biopsy for noninvasive prenatal testing (NIPT)[4,5,6] and for cancer management such as diagnosis, prognostication, treatment selection, and monitoring of the disease burden[3,7,8,9]
The cell-free DNA (cfDNA) assay in clinical fields has become possible with the advance in molecular diagnostic techniques, such as droplet digital polymerase chain reaction[10,11,12], next-generation sequencing (NGS)[13,14,15], and genome-wide sequencing[16,17]

Summary

Introduction

Nucleic acid-based molecular diagnosis is a key technology for implementing precision medicine in clinics[1,2,3]. CfDNA is a demanding analyte owing to its considerably low concentration in plasma, and it has not been widely adopted for clinical diagnosis and research[9,18]. Circulating tumor DNAs (ctDNAs) account for a fraction as low as 0.01% among the total cfDNA, where its concentration would be below the limit of detection for cancer diagnosis[13,25,26,27,28]. The spin column-based solid phase extraction method is frequently applied to quickly and extract nucleic acids[18,30,31]. This method is basically performed with a bench top centrifuge, which requires extremely high www.nature.com/scientificreports/. The troublous centrifugation process has to be repeated several times for the washing, drying, and eluting processes

Methods

Results

Conclusion