Rapid isolation of fungal genomic DNA suitable for long distance PCR.
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.
Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure links 653, Gent, 9000, Belgium. Sofie.DeMaeseneire@UGent.be
S L
SL
De Maeseneire
I N
IN
Van Bogaert
T
T
Dauvrin
W K
WK
Soetaert
E J
EJ
Vandamme
D001233:Aspergillus nidulans / Q000235:genetics; D004271:DNA, Fungal / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D016681:Genome, Fungal; D025282:Mycelium / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
D004271:DNA, Fungal