Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi.

By Jae-Hyuk Yu, Z. Hamari, Kap-Hoon Han, Jeong-Ah Seo, Y. Reyes-Domínguez, C. Scazzocchio. Fungal Genetics and Biology, 2004

1,155 citations recorded by the source corpus.

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.

Subfields: Biomedical Engineering, Insect Science, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/e361b00a2a1b635dd8b9c2d96d394e456c0b54b2

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Citations in the corpus

A Versatile and Efficient Gene-Targeting System for Aspergillus nidulans (2006, methodology): “These approaches have been facilitated by the development of fusion PCR (Kuwayama et al. 2002; Yang et al. 2004; Yu et al. 2004).”