Borges D B, Amorim M B, Waldschmidt A M, Mariano-Neto E, Vivas C V, Pereira D G
Departamento de Ciências Biológicas, Universidade Estadual do Sudoeste da Bahia, Jequié, BA, Brasil.
Genet Mol Res. 2012 May 22;11(2):1586-91. doi: 10.4238/2012.May.22.8.
Melanoxylon brauna (Fabaceae - Caesalpinioideae) is an endemic and valuable hardwood tree species in the Brazilian Atlantic rainforest; it is comparable to African ebony wood. We tested three protocols of DNA extraction based on the citrimonium bromide (CTAB) method and evaluated the quantity, purity and integrity of the DNA. We also determined whether these procedures interfere with PCR amplification in order to develop a protocol for M. brauna. We found that the quality and integrity of DNA were improved with the use of proteinase K in the extraction buffer and by modifications in the centrifugation speed. The lowest concentration of DNA was obtained with Doyle and Doyle's protocol (5.42 ng/μL). Ferreira and Grattapaglia's protocol modified for M. brauna provided the most DNA (36.89 ng/μL) and the highest quality DNA (purity ratio of 1.80 nm). The original Ferreira and Grattapaglia protocol provided 13.42 ng/μL DNA; however, the purity ratio (1.44 nm) indicates protein contamination. PCR results showed that Ferreira and Grattapaglia's protocol modified for M. brauna gave satisfactory quantity and purity of DNA for molecular studies.
黑木豆（豆科 - 云实亚科）是巴西大西洋雨林中的一种特有且珍贵的硬木树种；它可与非洲乌木相媲美。我们基于十六烷基三甲基溴化铵（CTAB）法测试了三种DNA提取方案，并评估了DNA的数量、纯度和完整性。我们还确定了这些程序是否会干扰聚合酶链式反应（PCR）扩增，以便为黑木豆开发一种方案。我们发现，在提取缓冲液中使用蛋白酶K并改变离心速度可提高DNA的质量和完整性。采用多伊尔和多伊尔的方案获得的DNA浓度最低（5.42纳克/微升）。为黑木豆修改后的费雷拉和格拉塔帕利亚的方案提供的DNA最多（36.89纳克/微升）且DNA质量最高（纯度比为1.80纳米）。费雷拉和格拉塔帕利亚的原始方案提供了13.42纳克/微升的DNA；然而，纯度比（1.44纳米）表明存在蛋白质污染。PCR结果表明，为黑木豆修改后的费雷拉和格拉塔帕利亚的方案为分子研究提供了数量和纯度均令人满意的DNA。
