Riahi M, Zarre S, Maassoumi A A, Attar F, Kazempour Osaloo S
Department of Plant Sciences, School of Biology, College of Science, University of Tehran, Tehran, Iran.
Genet Mol Res. 2010 Jul 13;9(3):1334-42. doi: 10.4238/vol9-3gmr839.
Three DNA extraction protocols were compared for their ability to yield DNA from the leaves of herbarium specimens of nine species from nine genera of the Papilionoideae. We tested two protocols that use classic procedures for lysis and purification with cetyl trimethylammonium bromide (CTAB); a third protocol used a Nucleospin Plant kit. DNA obtained from all three procedures was quantified and tested by PCR. Test results indicated the superiority of one of the CTAB protocols. We made some modifications, developing a protocol that produced high-quality DNA from all nine species. The modification involved the use of a lower EDTA concentration (20 mM instead of 50 mM) and a higher beta-mercaptoethanol concentration (1% instead of 0.4%) in the extraction buffer. The modified protocol avoids the necessity for a second DNA precipitation step. This new CTAB protocol includes the use of 1.4 M NaCl, 20 mM EDTA and 1% beta-mercaptoethanol in the extraction; DNA precipitation time is reduced. A reduction in contaminating metabolites (such as PCR inhibitors) in the sample mixtures and lower costs for reagents are characteristics of this modified protocol; the cost of analysis per sample was lowered, compared to previous options. The quality of DNA was suitable for PCR amplification. This is a practical alternative to more difficult, time-consuming and expensive protocols.
比较了三种DNA提取方法从蝶形花亚科九个属的九个物种的腊叶标本叶片中提取DNA的能力。我们测试了两种使用十六烷基三甲基溴化铵（CTAB）进行经典裂解和纯化程序的方法；第三种方法使用了植物核酸提取试剂盒。对从所有三种方法获得的DNA进行定量并通过PCR进行检测。测试结果表明其中一种CTAB方法具有优越性。我们进行了一些改进，开发出一种能从所有九个物种中提取高质量DNA的方法。改进之处在于在提取缓冲液中使用较低浓度的EDTA（20 mM而不是50 mM）和较高浓度的β-巯基乙醇（1%而不是0.4%）。改进后的方法无需进行第二次DNA沉淀步骤。这种新的CTAB方法在提取过程中使用1.4 M NaCl、20 mM EDTA和1%β-巯基乙醇；DNA沉淀时间缩短。样品混合物中污染性代谢物（如PCR抑制剂）减少以及试剂成本降低是这种改进方法的特点；与之前的方法相比，每个样品的分析成本降低。DNA质量适合PCR扩增。这是一种比更困难、耗时且昂贵的方法更实用的替代方法。
