Adaptation of a PCR-based cloning method for use in filamentous fungi

Michael Nielsen, Uffe H Mortensen, and Alexei Aleksenko

Author address: 

Technical University of Denmark, BioCentrum, Kgs. Lyngby, Denmark

Abstract: 

The rapid accumulation of genomic sequences from a broad range of organisms fuels the need for specific genome manipulation. A cloning-free PCR based method for allele replacement exists where PCR products are fused together to produce two tailored DNA fragments suitable for co-transformation and subsequent integration into the genome of a given host. The fusion is accomplished by the use of adaptamers, which are PCR primers with overhangs that differentially tag the 5' and 3' of the amplified substrate. Complimentary adaptamers of two denatured PCR fragments anneal, thus fusing the products prior to PCR amplification of the whole fragment. Using this technique one fragment is generated that contains sequences matching the desired site of integration fused to the 5' 2/3 of a selectable gene and another where the integrative sequences are fused to the 3' 2/3 of the same selectable gene. After transformation, homologous recombination in the cell fuses the two fragments to reconstruct the entire selectable marker and inserts the fragments into the genome at the desired site. If a counter selectable marker is used, it can be excised from the genome by a direct repeat recombination event, leaving only the desired genomic alteration. So far this technique only been used in Saccharomyces cerevisiae because its genomic sequence is known, but as more genomic sequences become available the approach may become applicable to other organisms. To demonstrate this, we have adapted the method for use in the filamentous fungus, Aspergillus nidulans, by using it to replace the wild type yA allele with a mutant allele
2001

abstract No: 

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Full conference title: 

21st Fungal Genetics Conference
    • Fungal Genetics Conference 21st (2000)