Faculty of Science, Sichuan Agricultural University, Yaan 625014, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Jan 15;881-882:63-8. doi: 10.1016/j.jchromb.2011.11.042. Epub 2011 Dec 6.
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.
尿液来源的人类基因组 DNA（gDNA）在临床医学、运动和法医学等多个领域具有广泛的应用。我们描述了一种使用羧基化磁性纳米颗粒（CMNPs）作为固相吸附剂从尿液样本中提取 gDNA 的新方法。尿液样本的沉淀与冷冻会显著减少 CMNPs 对细胞的捕获。然而，加入 10 mM EDTA 并随后调整 pH 值（pH 6.0-7.1）可以重新溶解尿液沉淀物。纯化的 gDNA 大小范围约为 0.1 kb 至 23 kb 以上。使用针对 K-ras、GAPDH、CYP3A4 和 GDF5 的特异性引物进行的 PCR 扩增了不同大小的基因片段，证明了所分离 DNA 的高质量。成功地展示了使用小至 50 μl 的尿液样本分离的 DNA 进行 PCR 扩增。尿液细胞的富集和随后的 DNA 吸附可以使用相同的 CMNPs 实现，大大简化了提取程序。CMNP gDNA 提取技术简单、快速、灵敏且环保，适用于常规实验室使用，并且有可能应用于自动化尿液提取平台。
