Moreira P A, Oliveira D A
Departamento de Biologia Geral, Ecologia Evolutiva e Biodiversidade, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.
Genet Mol Res. 2011 Mar 1;10(1):353-8. doi: 10.4238/vol10-1gmr1030.
Isolation of high-quality DNA from plants, especially plants from the Cerrado, is notoriously difficult because of polysaccharides and secondary compounds produced by plants from this biome. DNA isolation and its quality may be compromised by chemical defenses such as tannins and phenols. Quantitative plant defenses tend to have a cumulative effect, increasing in concentration during leaf development, reducing DNA quality extracted in mature compared to young leaves. We report the effect of leaf age on DNA extraction of Dimorphandra mollis. Our working hypothesis was that the young leaves have more DNA than old leaves of the same individual because chemical defenses accumulate in older leaves. Young and old leaves were sampled from eight mature trees as well as leaves from eight seedlings in the north region of Minas Gerais State. Genomic DNA extraction followed the standard CTAB procedure. DNA isolation was very successful from young leaves of 16 individuals of D. mollis. The extracted DNA exhibited high quality and the DNA quantity was also high, with an A(260)/A(280) ratio above 1.8, which is within the optimal sample range. In contrast, DNA isolation from old leaves was not successful. When the DNA was extracted from old leaves, the DNA was brownish, indicating contamination by phenolic compounds. These metabolites oxidize the DNA irreversibly, which hinders amplification of DNA by PCR by inhibiting the action of enzymes such as Taq polymerase. PCR performed with DNA from young leaves of D. mollis was successful and produced strong bands for RAPD markers.
从植物中,尤其是从塞拉多地区的植物中分离高质量的DNA非常困难,因为来自这个生物群落的植物会产生多糖和次生化合物。DNA的分离及其质量可能会受到单宁和酚类等化学防御物质的影响。植物的定量防御往往具有累积效应,在叶片发育过程中浓度会增加,与幼叶相比,成熟叶片中提取的DNA质量会降低。我们报告了叶龄对柔毛双蕊苏木DNA提取的影响。我们的工作假设是,同一植株的幼叶比老叶含有更多的DNA,因为化学防御物质会在老叶中积累。从米纳斯吉拉斯州北部地区的八棵成熟树木以及八株幼苗上采集了幼叶和老叶。基因组DNA提取遵循标准的CTAB程序。从16株柔毛双蕊苏木的幼叶中成功分离出了DNA。提取的DNA质量很高,DNA数量也很多,A(260)/A(280)比值高于1.8,处于最佳样本范围内。相比之下,从老叶中分离DNA没有成功。从老叶中提取DNA时,DNA呈褐色,表明受到了酚类化合物的污染。这些代谢产物会不可逆地氧化DNA,通过抑制Taq聚合酶等酶的作用来阻碍DNA的PCR扩增。用柔毛双蕊苏木幼叶的DNA进行PCR很成功,并产生了用于RAPD标记的强条带。