A Cost-Effective, Kit-Free Method for Extracting Nuclear and Mitochondrial DNA From Low-Abundance Cells in Urine Samples.
4 affiliations
- 1Department of Medical Technology and Sciences, School of Health Sciences at Narita, International University of Health and Welfare, Narita, JPN.
- 2Department of Medical Technology, Faculty of Medical and Health Sciences, Tsukuba International University, Tsukuba, JPN.
- 3Department of Clinical Laboratory Science, Faculty of Health Sciences, Japan Healthcare University, Sapporo, JPN.
- 4Department of Medical Laboratory Sciences, Graduate School, International University of Health and Welfare, Narita, JPN.
Abstract
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.
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BibTeX
@article{yamaguchi2026costeffective,
title = {{A Cost-Effective, Kit-Free Method for Extracting Nuclear and Mitochondrial DNA From Low-Abundance Cells in Urine Samples.}},
author = {Yoshitaka Yamaguchi and Koichi Yamaguchi and Hiroshi Suzuki and Junko Nomoto and Hironori Katayama},
journal = {Cureus},
year = {2026},
month = sep,
doi = {10.7759/cureus.116804},
url = {https://europepmc.org/article/MED/42927000},
note = {CC BY},
}
RIS
TY - JOUR
TI - A Cost-Effective, Kit-Free Method for Extracting Nuclear and Mitochondrial DNA From Low-Abundance Cells in Urine Samples.
AU - Yoshitaka Yamaguchi
AU - Koichi Yamaguchi
AU - Hiroshi Suzuki
AU - Junko Nomoto
AU - Hironori Katayama
PY - 2026
DA - 2026/09/23
JO - Cureus
DO - 10.7759/cureus.116804
UR - https://europepmc.org/article/MED/42927000
AB - 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.
SN - 2168-8184
ER -