Vazquez-Angulo J C, Mendez-Trujillo V, González-Mendoza D, Morales-Trejo A, Grimaldo-Juarez O, Cervantes-Díaz L
Instituto de Ciencias Agrícolas, Universidad Autónoma de Baja California, Baja California, México.
Genet Mol Res. 2012 May 15;11(2):1379-84. doi: 10.4238/2012.May.15.8.
Extraction of high-quality genomic DNA for PCR amplification from filamentous fungi is difficult because of the complex cell wall and the high concentrations of polysaccharides and other secondary metabolites that bind to or co-precipitate with nucleic acids. We developed a modified sodium dodecyl sulfate/phenol protocol, without maceration in liquid nitrogen and without a final ethanol precipitation step. The A(260/280) absorbance ratios of isolated DNA were approximately 1.7-1.9, demonstrating that the DNA fraction is pure and can be used for analysis. Additionally, the A(260/230) values were higher than 1.6, demonstrating negligible contamination by polysaccharides. The DNA isolated by this protocol is of sufficient quality for molecular applications; this technique could be applied to other organisms that have similar substances that hinder DNA extraction. The main advantages of the method are that the mycelium is directly recovered from culture medium and it does not require the use of expensive and specialized equipment.
从丝状真菌中提取用于PCR扩增的高质量基因组DNA很困难，这是因为其细胞壁结构复杂，且多糖和其他次生代谢产物浓度高，这些物质会与核酸结合或共沉淀。我们开发了一种改良的十二烷基硫酸钠/苯酚方法，无需在液氮中研磨，也无需最后的乙醇沉淀步骤。分离出的DNA的A(260/280)吸光率约为1.7 - 1.9，表明DNA组分纯净，可用于分析。此外，A(260/230)值高于1.6，表明多糖污染可忽略不计。通过该方法分离的DNA质量足以用于分子应用；该技术可应用于其他含有类似阻碍DNA提取物质的生物体。该方法的主要优点是菌丝体可直接从培养基中回收，且无需使用昂贵的专业设备。
