Primer Designer for Site-Directed Mutagenesis Alexey Novoradovsky 1, Vivian Zhang, Madhushree Ghosh 2, Holly Hogrefe 2, William Detrich 2, Joseph A. Sorge 2, Terry Gaasterland 1. INTRODUCTION QuikChange® Primer Designer is a part of the Stratagene LabTools website: This software designs efficient primers for site-directed mutagenesis by calculating free energy of the mismatched primer-template duplex and comparing it to the energy of perfect duplex. The result of such comparison is interpreted as an energy cost of a mismatch. Preference in primer design strategy is directed toward minimizing of the energy cost, which also results in fewer nucleotide substitutions or result in mismatches capable of base-pairing (e.g. G/T). In addition to energy conservation rules, we analyzed data obtained with degenerate primers and incorporated several empirical rules regarding optimal codon replacement, which were also included in this software. METHODS Codon Selection: If multiple codon choices are possible then the preference is made in favour of fewer base changes. For example, if threonine encoded by ACG is to be mutated to arginine then the desired change can be performed by six different ways: AGA, AGG, CGA, CGC, CGG, or CGT. However the program would recommend AGG as a target, because it only involves a single base change. Further improvements of the codon selection were introduced by the results of SDM experiments with the degenerate primers. In these experiments, primers contained a single degenerated codon NNK (which includes 32 possible triplets, encoding for 19 amino acids: ACDEFGHILMNPQRSTVWY). The numbers of resulted mutant DNA sequences were counted among 607 mutants, and the codon biases were calculated as a deviation of the observed number of resulting codons (NO) from expected 1/32 of the total number of analyzed mutants (NE). The codon biases ranged from -12 to +23 (Table 1). Table 1. Codon biases revealed by the SDM experiments with degenerate primers. Codons with the highest positive biases contain significantly higher proportions of dinucleotides AG, AT, AA, TA, AC, and GA, while dinucleotides CT, TG, TT, CC, and GT were underrepresented among observed mutants. These observations are also considered by the codon selection program. Primer Design: Free energy of a primer-template duplex was calculated as a sum of stacking energies of nearest neighbours. Mismatches in a duplex were treated as the internal loops or bulges, flanked by perfect duplex branches. The calculation principles derived from the nearest- neighbour energy calculation of nucleic acid duplex (Nielsen et al, 1995, SantaLucia et al.,1996). They are based on summarizing the nearest dinucleotide-duplex energies, where the members of the tetrads can be either parts of perfectly matched branch duplexes or the internal loops/bulges or parts of terminal dangling ends. Web interface: Primer design process is piped through the submission of HTML forms with generating and passing variables via PHP scripts. Registered SDM primer designer users are validated through the PostgreSQL user database containing usernames and encrypted passwords. All possible matched or unmatched nucleotide tetrads at temperatures from 37 o C to 85 o C are stored in another PostgreSQL database, which is accessed through PHP postgres functions. All the calculations are performed through PHP scripts. ABSTRACT Engineering mutations within cloned DNA fragments involves the design of primer- template duplexes containing the target mutation. These primers have single or multiple base mismatches, or in the case of a deletion or an insertion, single-stranded DNA loops. Such DNA duplexes have a higher free energy than a mismatch-free perfect duplex. Our program suggests the most energy-saving, and thus the most effective, base substitutions to generate single or multiple amino acid changes, frame-shifts, deletions, or insertions within the target DNA molecules. Preference in codon replacement is given to the codon changes that involve fewer nucleotide substitutions and are more energetically favorable. The energy cost is calculated as a difference between the summary stacking and nearest-neighbor energies of the perfect primer-template duplex compared to the duplex with mismatches. In addition to free energy conservation principles, the software incorporates several empirical rules for nucleotide changes, which were established through mutagenesis experiments using degenerate primers. The program is written in PHP and is accessible as a free web service in the Stratagene web site: 1. Select program. DISCUSSION The example in Figure 1 demonstrates one of the four available primer design routines, single amino acid change. Other applications, that are not illustrated here, include: 1) optimization of codon selection, described in Methods; 2) design of single or multiple primers with up to five single-nucleotide changes; 3) design of multiple mutagenesis primers for simultaneous introduction of up to five amino acid changes in one experiment, implemented by Stratagenes QuikChange® Multi kit; and 4) design of primers to generate deletions or insertions in the target DNA sequence. Further information about the Primer Designer for Site-Directed Mutagenesis can be obtained through the help hyperlinks available to the registered users. Registration for this service, as well as for other free Stratagene LabTools services, can be obtained at Primer Designer for Site-Directed Mutagenesis service was launched in July 2003, and during the last eight months the user database includes about 7,000 users. CONCLUSIONS 1. Primer design for site-directed mutagenesis was addressed through integration of primer-template duplex energy calculation with the data on codon biases established through SDM experiments with degenerate primers. 2. PHP-enabled web interface has been created to provide user access to the database- driven server-side applications. REFERENCES 1. Nielsen, D.A., Novoradovsky, A., Goldman, D. SSCP primer design based on single-strand DNA structure predicted by a DNA folding program. Nucleic Acid Research, 1995, 23: SantaLucia, J. Jr., Allawi, H.T., Seneviratne, P.A. Improved nearest- neighbor parameters for predicting DNA duplex stability. Biochemistry, 1996, 35: Otherwise, the untranslated DNA sequence will be displayed; 3, amino acid change must be specified by checking the radio button in the gray area and selecting the target amino acid (or termination codon) from the drop-down menu; 4a, recommended sense and antisense primers with melting temperature close to 80 o C are shown in the table. The associated free energy of the mismatched primer-template duplexes, and the energy costs associated with the suggested are also displayed. Note: the Designer automatically suggests the most energy-cost-effective codon AGG as requiring the only base change, and containing good dinucleotide AG (see METHODS). The hyperlink to template sequence at the top of the page can be used to view the position of the designed primer within DNA template. Figure 1. The example of using QuikChange® Primer Designer to design a single SDM primer for mutating Methionine-77 to Arginine. 1, program selection can be one using the left-side menubar or using the hyperlinks in the page body; 2, DNA sequences can be entered as flat-text FASTA or GenBank-formatted files either using the file control or directly by copying-pasting to the provided textarea. If amino acid changes are to be performed then the translated sequence can be displayed at the next step either completely or within specified translation range. 2. Using available options, enter DNA sequence, its format and translation range. 4a. View designed sense and antisense SDM primers and their characteristics. 4b. (Optional): view position of the designed primer within DNA template. 3. Define amino acid change and annealing temperature. 1. The Rockefeller University, New York, NY Stratagene Cloning Systems, La Jolla, CA 92037