title: PCR-ready human DNA extraction from urine samples using magnetic nanoparticles.
abstract: Urine-derived human genomic DNA (gDNA) has wide application in a variety of disciplines including clinical medicine, sports, and forensic science. We describe a novel method for gDNA extraction from urine samples using carboxylated magnetic nanoparticles (CMNPs) as solid-phase adsorbents. Sedimentation associated with freezing of urine samples significantly reduces cell capture by CMNPs. However, the addition of 10 mM EDTA and subsequent pH modification (pH 6.0-7.1) can re-dissolve urine sediments. Purified gDNA ranged from around 0.1 kb to more than 23 kb. PCR using specific primers targeting K-ras, GAPDH, CYP3A4 and GDF5 amplified 100% of varying sized gene fragments, verifying the high quality of the isolated DNA. Successful PCR amplifications using DNA isolated from urine samples as small as 50 μl were demonstrated. Enrichment of urine cells and subsequent adsorption of DNA can be achieved with the same CMNPs, greatly simplifying extraction procedures. The CMNP gDNA extraction technique proved to be simple, rapid, sensitive and environmentally friendly, with application for routine laboratory use and potentially within automated urine extraction platforms.
authors.affiliation: Faculty of Science, Sichuan Agricultural University, Yaan 625014, China. mesopig@hotmail.com
authors.forename: Zhi
authors.initials: Z
authors.lastname: Shan
authors.forename: Zhongwu
authors.initials: Z
authors.lastname: Zhou
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Zhang
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhou
authors.forename: Anxiang
authors.initials: A
authors.lastname: Wen
authors.forename: Ken D
authors.initials: KD
authors.lastname: Oakes
authors.forename: Mark R
authors.initials: MR
authors.lastname: Servos
mesh_terms: D000327:Adsorption; D000328:Adult; D005591:Chemical Fractionation / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification* / Q000652:urine; D004587:Electrophoresis, Agar Gel; D005260:Female; D006801:Humans; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D008297:Male; D008854:Microscopy, Electron; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D058185:Magnetite Nanoparticles; D004247:DNA