A Cost-Effective, Kit-Free Method for Extracting Nuclear and Mitochondrial DNA From Low-Abundance Cells in Urine...

This study evaluates a simplified, cost-effective DNA extraction protocol using only 10% sodium chloride (NaCl) and Proteinase K (ProK) to isolate both nuclear DNA (nuDNA) and mitochondrial DNA (mtDNA) from low-abundance cells in human urine, providing an open-source alternative to expensive commercial kits. Urine samples from six healthy participants were centrifuged, washed with phosphate-buffered saline, lysed with the NaCl/ProK mixture at 60°C overnight, and recovered via ethanol precipitation. To validate downstream applicability, polymerase chain reaction (PCR) amplification was performed targeting the nuclear FGFR3 (423 bp) and the mitochondrial D-loop regions (HV-1: 634 bp, HV-2: 520 bp), followed by agarose gel electrophoresis and Sanger sequencing. The extracted DNA concentrations ranged from undetected to 146.0 μg/mL. PCR amplification of the nuclear FGFR3 succeeded in five out of six samples (83.3%), while the mitochondrial regions (HV-1 and HV-2) achieved a 100% success rate, yielding robust bands even in the sample with undetectable initial genomic DNA. Sanger sequencing and Basic Local Alignment Search Tool search confirmed 100% (FGFR3), 99.4% (HV-1), and 97.8% (HV-2) homology with reference sequences (National Center for Biotechnology Information: NC_012920.1). In conclusion, sequence-ready nuDNA and mtDNA can be successfully extracted using this minimal-reagent, kit-free method. The significantly higher sensitivity of mtDNA highlights its potential for non-invasive genetic diagnostics and point-of-care liquid biopsy in low-biomass specimens.