Computational Principles of Primer Design for Site Directed Mutagenesis

Site directed mutagenesis (SDM) is a method to alter DNA molecules with intentionally designed mutations. SDM creates mutated copies of the original sequence as follows: an oligonucleotide primer hybridizes to the DNA template to form a duplex with base mismatches or singlestranded DNA loops. Then, DNA polymerase extends the primer to create a mutated copy of the initial DNA molecule. In this work, we address the problem of designing optimal 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 substitutions, preference in codon replacement is given to changes that minimize the number of nucleotide substitutions. Second, codons vary in the frequency with which they are incorporated into successful mutants, so highly successful codons are favored. We conducted a series of SDM experiments to determine success rates for all codons. These rates are used in primer design. Third, primer length and primer position relative to the mutated site is chosen to minimize duplex free energy, and thus maximize duplex stability.