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Background  We developed a method to make a various high quality random peptide libraries for evolutionary protein engineering based on a combinatorial DNA synthesis.    Results  A split synthesis in codon units was performed with mixtures of bases optimally designed by using a Genetic Algorithm program. It required only standard DNA synthetic reagents and standard DNA synthesizers in three lines. This multi-line split DNA synthesis (MLSDS) is simply realized by adding a mix-and-split process to normal DNA synthesis protocol. Superiority of MLSDS method over other methods was shown. We demonstrated the synthesis of oligonucleotide libraries with 10 16  diversity, and the construction of a library with random sequence coding 120 amino acids containing few stop codons.    Conclusions  Owing to the flexibility of the MLSDS method, it will be able to design various "rational" libraries by using bioinformatics databases.

Multi-line split DNA synthesis: a novel combinatorial method to make high quality peptide libraries.