Isolation of urinary cell-free DNA using agarose gel-gate transient isotachophoresis

Cell-free DNA (cfDNA) is DNA found in complex body fluids such as plasma and urine and receives significant interest in liquid biopsy. However, the majority of cfDNA fragments for diagnosis are short (≤200 bp), thus it is difficult to extract them by solid-phase extraction, a commonly used method of DNA extraction. We have developed an open fluidic electrophoresis device that can partition a channel with multiple types of agarose gels, allowing easy placement of different gel compartments in a single electrophoretic path and enabling stable transient isotachophoresis (tITP) by suppressing the diffusion of each electrolyte between compartments. We demonstrated that Mycobacterium tuberculosis (MTB) genome DNA fragments ranging from 100–200 could be successfully isolated by tITP from plasma after a simple deproteinization. Furthermore, by adding a simple pre-adsorption process using anion exchange group-modified beads, we achieved selective separation of 45 bp short-strand MTB DNA fragments from about 10 ml of urine, thereby improving the sensitivity of short cfDNA detection with PCR.