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Optimization of a genome‐walking method to suit GC‐rich template DNA from biotechnological relevant Actinobacteria

SiteFinding‐PCR has been recently reported to be a useful technique in order to identify unknown DNA fragments located adjacent to available sequences. However, this method has so far only been applied to few DNA sources including plants, samples from bioleaching communities, and a Pseudomonas strain. In order to complete the sequence information of two gene clusters in Gram‐positive rhodococci the original protocol was applied yielding amplicons of insufficient size. The binding site of the previously published SiteFinder‐2 oligo proved to be unsuitable for Rhodococcus and other members of the Actinobacteria since the binding motif occurred too frequently. Available genome sequences of different Actinobacteria were analysed and the binding site of the SiteFinder oligo modified. Moreover, PCR conditions were adapted to the high GC content of the template DNA allowing the successful adaptation of this method to two members of the Actinobacteria. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)