Arruda S R, Pereira D G, Silva-Castro M M, Brito M G, Waldschmidt A M
Programa de Pós-Graduação em Genética, Biodiversidade e Conservação, Departamento de Ciências Biológicas, Universidade Estadual do Sudoeste da Bahia, Jequié, BA, Brasil.
Departamento de Química e Exatas, Universidade Estadual do Sudoeste da Bahia, Jequié, BA, Brasil.
Genet Mol Res. 2017 Jul 6;16(3):gmr-16-03-gmr.16039063. doi: 10.4238/gmr16039063.
Some species are characterized by a high content of tannins, alkaloids, and phenols in their leaves. These secondary metabolites are released during DNA extraction and might hinder molecular studies based on PCR (polymerase chain reaction). To provide an efficient method to extract DNA, Mimosa tenuiflora, an important leguminous plant from Brazilian semiarid region used in popular medicine and as a source of fuelwood or forage, was used. Eight procedures previously reported for plants were tested and adapted from leaf tissues of M. tenuiflora stored at -20°C. The optimized procedure in this study encompassed the utilization of phenol during deproteinization, increased concentrations of cetyltrimethylammonium bromide and sodium chloride, and a shorter period and lower temperature of incubation concerning other methods. The extracted DNA did not present degradation, and amplification via PCR was successful using ISSR, trnL, ITS, and ETS primers. Besides M. tenuiflora, this procedure was also tested and proved to be efficient in genetic studies of other plant species.
一些物种的叶子富含单宁、生物碱和酚类物质。这些次生代谢产物在DNA提取过程中会释放出来，可能会妨碍基于PCR（聚合酶链反应）的分子研究。为了提供一种高效的DNA提取方法，本研究使用了含羞草（Mimosa tenuiflora），这是一种来自巴西半干旱地区的重要豆科植物，在传统医学中使用，也是薪柴或饲料的来源。对先前报道的八种植物提取程序进行了测试，并根据储存在-20°C的含羞草叶片组织进行了调整。本研究中的优化程序包括在脱蛋白过程中使用苯酚、增加十六烷基三甲基溴化铵和氯化钠的浓度，以及与其他方法相比更短的孵育时间和更低的孵育温度。提取的DNA没有降解，使用ISSR、trnL、ITS和ETS引物通过PCR成功扩增。除了含羞草外，该程序还在其他植物物种的遗传研究中进行了测试，并被证明是有效的。
