Whole genome amplification from filamentous fungi using Phi29-mediated multiple displacement amplification.
The availability of genomic DNA of sufficient quality and quantity is fundamental to molecular genetic analysis. Many filamentous fungi are slow growing or even unculturable and current DNA isolation methods are often unsatisfactory. We have used multiple displacement amplification (MDA) to amplify whole genomes for two fungal species, Penicillium paxilli and the slow growing endophyte of grasses Epichloe festucae. Up to 10 microg of high molecular weight DNA was routinely amplified from less than 10 ng of template DNA obtained using glass bead-mediated disruption of fungal spores or alkaline lysis of mycelium. PCR was possible from MDA-generated DNA and amplicons up to 10 kb were successfully amplified. RFLP analysis was successful, with bands of up to 5 kb routinely detected. Hybridization of MDA-amplified DNA to a cosmid library illustrated that the MDA product amplified from E. festucae is representative of the genome. MDA is a reliable method that could be applied to applications ranging from high-throughput screening of deletion mutants to genomic library construction.
Foster
Simon J
SJ
Centre for Functional Genomics, Institute of Molecular BioSciences, Massey University, Private Bag 11 222, Palmerston North 5321, New Zealand. s.j.foster@massey.ac.nz
Monahan
Brendon J
BJ
D017103:Bacillus Phages / Q000201:enzymology*; D004271:DNA, Fungal / Q000302:isolation & purification / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D016681:Genome, Fungal; D025301:Hyphae / Q000235:genetics; D006999:Hypocreales / Q000235:genetics*; D008970:Molecular Weight; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D010407:Penicillium / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D013172:Spores, Fungal / Q000235:genetics
D004271:DNA, Fungal; D004259:DNA-Directed DNA Polymerase