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Creative Biostructure has extensive expertise in custom mutagenesis libraries services using DNA shuffling method. Our DNA shuffling technology can offer plenty of gene diversification for directed evolution.
DNA shuffling is a method of in vitro recombination to rapidly propagate different DNA mutations from different species. This technique depends on the homologous gene fragments can be randomly recombined by DNaseI or restriction enzymes. Up to now, DNA shuffling is a powerful tool used to increase mutagenesis librari size.
Scientists from Creative Biostructure has strong expertise in performing excellent procedures to obtain DNA shuffling:
1. Acquiring DNA fragments for shuffling
a. Getting parent DNA for shuffling;
b. Purifying PCR products, the DNA concentration after purification should be at least 40 μg/mL;
c. Digesting parental genes using DNaseI to generate a pool of fragments;
d. Purifying the desired DNA fragment size on an agarose gel.
2. Reassembly of DNaseI fragments
Adding purified DNA fragments with Pfu enzymes to run the PCR reaction cycle.
3. Aamplificaiton of full-length sequences
Annealing and extending the fragment DNA using appropriate primers. Using this method, we can obtain a number of mutagenesis libraries with high mutagenesis rate.
Most notably, DNA shuffling can also be generated using restriction enzymes, in which DNA fragments will be jointed with DNA ligase.
Figure 1. Schematic of DNA shuffling process.
Besides DNA shuffling, Creative Biostructure provides other in vitro recombination strategies based on recombining properties coded in similar genes to generate mutagenesis libraries, such as random chimeragenesis on transient templates (RACHITT) and staggered extension process (StEP). RACHITT can employ no thermocycling, strand switching, or staggered extension, but rather the trimming, gap filling, and ligation of DNA fragments hybridized on a parental DNA template. While StEP is a modified PCR procedure in which the elongation step is interrupted prematurely by heat denaturation. Subsequently, annealing allows incomplete extension products to switch templates, effecting recombination of multiple DNA templates into one amplicon.
Creative Biostructure’s custom mutagenesis libraries provides you with a wide range of gene diversification for directed protein evolution. We also provide optimization of DNA shuffling for high fidelity recombination. Creative Biostructure provides a full range of protein engineering and analysis services for your specific projects.
Please feel free to contact us to discuss your project!
DNA shuffling. (https://en.wikipedia.org/wiki/DNA_shuffling)
J. M. Joern (2003). Directed Evolution Library Creation. Volume 231 of the series Methods in Molecular Biology™, pp 85-89.
W. M. Coco, et al. (2001). DNA shuffling method for generating highly recombined genes and evolved enzymes. Nature Biotechnol., 19: 354-359.