Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi.
Gene replacement via homologous double crossover in filamentous fungi requires relatively long (preferentially >0.5 kb) flanking regions of the target gene. For this reason, gene replacement cassettes are usually constructed through multiple cloning steps. To facilitate gene function studies in filamentous fungi avoiding tedious cloning steps, we have developed a PCR-assisted DNA assembly procedure and applied it to delete genes in filamentous fungi. While the principle of this procedure is essentially the same as other recently reported PCR-based tools, our technique has been effectively used to delete 31 genes in three fungal species. Moreover, this PCR-based method was used to fuse more than 10 genes to a controllable promoter. In this report, a detailed protocol for this easy to follow procedure and examples of genes deleted or over-expressed are presented. In conjunction with the availability of genome sequences, the application of this technique should facilitate functional characterization of genes in filamentous fungi. To stream line the analysis of the transformants a relatively simple procedure for genomic DNA or total RNA isolation achieving approximately 100 samples/person/day is also presented.
Department of Food Microbiology and Toxicology, The University of Wisconsin, Madison, WI 53706, USA. jyu1@wisc.edu
Jae-Hyuk
JH
Yu
Zsuzsanna
Z
Hamari
Kap-Hoon
KH
Han
Jeong-Ah
JA
Seo
Yazmid
Y
Reyes-Domínguez
Claudio
C
Scazzocchio
D001233:Aspergillus nidulans / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005800:Genes, Fungal; D016681:Genome, Fungal; D009492:Neurospora crassa / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D017931:DNA Primers; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins