An improved colony-PCR method for filamentous fungi for amplification of PCR-fragments of several kilobases.
A method was developed to perform PCR directly on mycelial pellets or colonies treated with NOVOzym 234. The method allows rapid screening of large numbers of transformants of both sporulating and non-sporulating fungi for the presence of (co)transforming plasmid copies or for specific genetic modifications such as gene disruption and site specific integration. PCR fragments of at least 3.2 kb can be obtained. Using this method the identification of specific disruption mutants from Aspergillus niger and Beauveria bassiana was carried out.
van Zeijl
C M
CM
TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene Technology, Zeist, The Netherlands.
van de Kamp
E H
EH
Punt
P J
PJ
Selten
G C
GC
Hauer
B
B
van Gorcom
R F
RF
van den Hondel
C A
CA
D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004271:DNA, Fungal / Q000235:genetics*; D004798:Enzymes / Q000494:pharmacology; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics
D004271:DNA, Fungal; D004798:Enzymes; C045154:novozym 234; D003577:Cytochrome P-450 Enzyme System; D005087:Glucan 1,4-alpha-Glucosidase