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Computational Principles of Primer Design for Site Directed Mutagenesis

A. Novoradovsky, V. Zhang, M. Ghosh, H. Hogrefe, J.A. Sorge and T. Gaasterland
The Rockefeller University, US

biotechnology, DNA Hybridization, computational methods, numerics

Site directed mutagenesis (SDM) is the process of altering a target DNA sequence by creating mutated copies of the original sequence where changes are intentionally designed. In the first step of an SDM experiment, an oligonucleotide primer hybridizes with the target DNA template to form a duplex that has one or more base mismatches or single-stranded DNA loops. At subsequent steps, DNA polymerase extends this imperfect DNA duplex to create a mutated copy of the initial molecule. SDM experiments tend to be more successful when using higher stability primer-template duplexes. In this work, we address the problem of optimizing primers to introduce single or multiple-site mutations, deletions, or insertions. Our approach is based on three principles. First, for mutations that introduce amino-acid changes within protein-coding DNA regions, preference in codon replacement is given to the changes that minimize the number of nucleotide substitutions. Second, heuristic rules for codon replacement have been established through SDM experiments with degenerate primers, in which different codons were observed with different frequencies among the successful mutant sequences. Third, primer length and primer position relative to the mutated site is chosen to minimize duplex free energy, thus maximizing duplex stability.

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