We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). Lehtonen, Soili I Taskinen, Barbara Ojala, Elina Kukkurainen, Sampo Rahikainen, Rolle RiihimÃ¤ki, Tiina A Laitinen, Olli H Kulomaa, Markku S HytÃ¶nen, Vesa PĮfficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub- cloning strategy for gene expression in suitable vectors/hosts.Įfficient preparation of shuffled DNA libraries through recombination (Gateway) cloning. Finally, we found several open reading frames (ORFs) with previously described potential medical application. The medium-large insert (7-15 kb) cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library however, the sequencing of clones one-by-one would be very time-consuming and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential however, unfortunately they would remain unexplored. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be 'domesticated' for downstream characterization and application, and this makes screening both laborious and expensive.

Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. Natural environments represent an incredible source of microbial genetic diversity. Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.ĭÅ¾unkovÃ¡, MÃ¡ria D'Auria, Giuseppe PÃ©rez-Villarroya, David Moya, AndrÃ©s

Serial cloner clone in oligo