Improving Random Mutagenesis by Purification of the Oligonucleotide Variants

Author(s): P. Gaytan, J. Yanez, R. Grande, E. Morett, X. Soberon

Journal Name: Combinatorial Chemistry & High Throughput Screening
Accelerated Technologies for Biotechnology, Bioassays, Medicinal Chemistry and Natural Products Research

Volume 8 , Issue 6 , 2005

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An oligonucleotide-based mutagenesis method is presented where, contrary to most classical mutagenic approaches, preselection of the variants is performed at the oligonucleotide level to avoid cloning of non-desired sequences. The method relies on the generation of differentially phosphate-protected oligonucleotides. Protection of the phosphates is accomplished by substoichiometric incorporation of an Fmoc-protected and n-propyl-protected trinucleotide phosphoramidite during ordinary oligonucleotide assembly. Instead of the alkali-labile β-cyanoethyl group introduced in ordinary assembly, the trinucleotide introduces the alkali-stable n-propyl group. As a result, single mutants carry three ionic phosphates less than the wild-type sequence, double mutants carry six ionic phosphates less and so on. This difference in ionic ratio enables separation of the variants by conventional polyacrilamide gel electrophoresis. In the exemplified library described herein, two sub-populations containing mainly triple and quadruple mutants were selected out of five possible sub-populations.

Keywords: Protein, directed evolution, mutagenesis method, codon-based, Fmoc, trinucleotide, libraries, alkylphosphotriester

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Article Details

Year: 2005
Page: [537 - 544]
Pages: 8
DOI: 10.2174/1386207054867355
Price: $65

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