De Maeseneire S L, Van Bogaert I N, Dauvrin T, Soetaert W K, Vandamme E J
Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure links 653, Gent, 9000, Belgium.
Biotechnol Lett. 2007 Dec;29(12):1845-55. doi: 10.1007/s10529-007-9483-6. Epub 2007 Aug 7.
A quick and reliable method for screening fungal transformants for specific genetic modifications is essential for many molecular applications. We have compared the applicability of a few rapid DNA extraction methods for Myrothecium and Aspergillus and tested the resulting DNA as to its suitability for PCR. For Myrothecium gramineum, the highest DNA concentration was obtained with the procedure described by N. Vanittanakom et al. (J Clin Microbiol 2002, 40: 1739-1742). For A. nidulans, concentrations higher than 100 ng/mul were reached with the glass bead, the LiCl, the boiling, the liquid N(2) and the protoplast-based method. Samples of M. gramineum resulting from the boiling and the liquid N(2) procedure were suitable for the amplification of fragments up to 2.3 kb. The direct use of mycelium from M. gramineum in the PCR tube can be employed for the reproducible amplification of fragments up to 1 kb. Amplification of fragments up to 4.3 kb requires the use of the Elongase Mix on samples extracted with the liquid N(2) procedure.
对于许多分子应用而言，一种快速可靠的筛选特定基因修饰真菌转化体的方法至关重要。我们比较了几种快速DNA提取方法对漆斑菌属和曲霉菌属的适用性，并测试了所得DNA用于PCR的适用性。对于禾本科漆斑菌，采用N. Vanittanakom等人（《临床微生物学杂志》2002年，40卷：1739 - 1742页）所述方法获得的DNA浓度最高。对于构巢曲霉，采用玻璃珠法、LiCl法、煮沸法、液氮法和原生质体法可使DNA浓度达到高于100 ng/μl。通过煮沸法和液氮法处理的禾本科漆斑菌样品适用于扩增长达2.3 kb的片段。直接将禾本科漆斑菌的菌丝体放入PCR管中可用于可重复地扩增长达1 kb的片段。扩增长达4.3 kb的片段需要对采用液氮法提取的样品使用延伸酶混合物。
