This study assessed three commercially available cell-free DNA (cfDNA) extraction kits and the impact of a PEG-based DNA cleanup procedure (DNApure) on cfDNA quality and yield. Six normal donor urine and plasma samples, and specimens from four pregnant (PG) women carrying male fetuses underwent extractions with the JBS cfDNA extraction kit (kit J), MagMAX Cell-Free DNA Extraction kit (kit M), and QIAamp Circulating Nucleic Acid Kit (kit Q). Recovery of a PCR product spike-in, endogenous TP53, and Y-chromosome DNA was used to assess kit performance. Nucleosomal-sized DNA profiles varied among the kits, with prominent multi-nucleosomal-sized peaks present in urine and plasma DNA isolated by kits J and M only. Kit J recovered significantly more spike-in DNA compared with kit M or Q (p<0.001) from urine, and similar amounts from plasma (p=0.12). Applying DNApure to kit M- and Q-isolated DNA significantly improved the amplification efficiency of spike-in DNA from urine (p<0.001) and plasma (p≤0.013). Furthermore, kit J isolated significantly more Y-chromosome DNA from PG urine compared to kit Q (p=0.05). We conclude that DNApure provides an efficient means of improving the yield and purity of cfDNA and minimizing effects of pre-analytical biospecimen variability on liquid biopsy assay performance.

中文翻译：

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提高尿液和无浆细胞DNA提取效率和纯度的新方法

这项研究评估了三种市售的无细胞DNA（cfDNA）提取试剂盒，以及基于PEG的DNA净化程序（DNApure）对cfDNA质量和产量的影响。使用JBS cfDNA提取试剂盒（试剂盒J），MagMAX无细胞DNA提取试剂盒（试剂盒M）和QIAamp循环核酸，提取了六份正常的供体尿液和血浆样品，以及四名携带男性胎儿的孕妇（PG）的标本。套件（套件Q）。PCR产品掺入，内源TP53和Y染色体DNA的回收用于评估试剂盒性能。试剂盒中核仁大小的DNA谱图各不相同，仅通过试剂盒J和M分离出的尿液和血浆DNA中存在明显的多核小体大小的峰。与试剂盒M或Q相比，试剂盒J从尿液中回收的刺入DNA明显多得多（p <0.001），从血浆中回收的刺入DNA量相似（p = 0.12）。将DNApure应用于试剂盒M和Q分离的DNA显着提高了从尿液（p <0.001）和血浆（p≤0.013）的掺入DNA的扩增效率。此外，与试剂盒Q相比，试剂盒J从PG尿液中分离出更多的Y染色体DNA（p = 0.05）。我们得出的结论是，DNApure提供了一种提高cfDNA产量和纯度并最大程度地减少分析前生物样本变异性对液体活检分析性能的影响的有效手段。