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Assembly of Designed Oligonucleotides as an Efficient Method for Gene Recombination: A New Tool in Directed Evolution
Author(s) -
Zha Dongxing,
Eipper Andreas,
Reetz Manfred T.
Publication year - 2003
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200390011
Subject(s) - directed evolution , dna shuffling , oligonucleotide , gene , recombination , bacillus subtilis , mutant , genetics , flp frt recombination , biology , lipase , computational biology , chemistry , genetic recombination , enzyme , biochemistry , bacteria
A new and practical method for gene recombination with formation of libraries of mutant genes is presented. The method is based on the assembly of appropriately prepared oligonucleotides whose design is guided by sequence information. High recombination frequency with formation of full‐length products is achieved by controlled overlapping of the designed oligomers. This process (ADO) minimizes self‐hybridization of parental genes, which constitutes a significant advantage over conventional family shuffling as used in the directed evolution of functional enzymes. ADO was applied to the recombination of two lipase family genes from Bacillus subtilis (LipA and LipB). In a library of 3000 lipase variants created by this method, several were found that display increased enantioselectivity in a model reaction involving the hydrolysis of a meso ‐diacetate.