School of Biosciences and Biotechnologies, Via Gentile III da Varano, University of Camerino, 62032 Camerino, Italy.
Mol Cell Probes. 2011 Feb;25(1):44-8. doi: 10.1016/j.mcp.2010.10.003. Epub 2010 Oct 26.
Long-term stored (LTS) whole blood collection can be an important source of DNA without collection costs, but there is a lack of information on methods useful to extract genomic DNA from such type of biological material. Here we report a simple and fast revisited phenol/chloroform extraction method from LTS whole blood. Protocol reliability was assessed by comparison with proteinase K and silica-gel membrane spin column-based DNA extraction methods using LTS -20 °C whole blood from 1980, and by testing it on 82 whole blood samples, collected from 1980 to 1995, with high quality (A(260/280) = 1.79 ± 0.32 O.D., A(260/230) = 1.45 ± 0.52 O.D.) and quantity results. Genotyping efficiency was also checked by performing RFLP-PCR and ASP-PCR of p53 Pro72Arg (rs1042522) SNP and hTERT MNS16A VNTR, respectively, resulting in 100% of samples successfully typed. In addition to the goodness and the efficiency of method proposed here, this protocol achieves working time reduction combining extraction and purification steps, allowing to work at room temperature. Furthermore, phenol is able to inactivate any potential nuclease and potential infective sources from the first step on. Based on these results we also conclude that LTS -20 °C whole blood samples may be considered a reliable and potential resource for future genotyping studies and retrospective analysis in a genetic epidemiological setting.
长期储存（LTS）全血采集可以作为一种无需采集成本的 DNA 重要来源，但缺乏有关从这种类型的生物材料中提取基因组 DNA 的有用方法的信息。在这里，我们报告了一种从 LTS 全血中提取基因组 DNA 的简单快速的重苯酚/氯仿提取方法。通过与 20°C 保存的 1980 年 LTS 全血的蛋白酶 K 和硅基膜离心柱法进行比较，以及对 1980 年至 1995 年采集的 82 份高质量（A(260/280) = 1.79 ± 0.32 OD，A(260/230) = 1.45 ± 0.52 OD）和高数量的全血样本进行测试，评估了该方案的可靠性。通过 p53 Pro72Arg(rs1042522) SNP 和 hTERT MNS16A VNTR 的 RFLP-PCR 和 ASP-PCR 分别检测基因分型效率，结果显示 100%的样本成功分型。除了该方法的有效性和效率之外，该方案还通过将提取和纯化步骤结合起来，实现了工作时间的缩短，允许在室温下进行操作。此外，从第一步开始，苯酚就能使任何潜在的核酸酶失活，并使潜在的感染源失活。基于这些结果，我们还得出结论，20°C 保存的 LTS 全血样本可被视为未来遗传流行病学研究和回溯分析中基因分型的可靠和潜在资源。
