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

摘要: Abstract 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 cassettes are usually constructed through multiple cloning steps. To facilitate function studies avoiding tedious steps, we have developed a PCR-assisted DNA assembly procedure and applied it to delete genes fungi. While principle is essentially same as other recently reported PCR-based tools, our technique has been effectively used 31 three fungal species. Moreover, method was fuse more than 10 controllable promoter. In report, detailed protocol for easy follow examples deleted or over-expressed presented. conjunction with availability genome sequences, application should functional characterization stream line analysis transformants simple genomic total RNA isolation achieving ∼100 samples/person/day also