##About RubyPrimer is a webtool for creating mutagenic oligonucleotide primers for use in the Quikchange II Site-Directed Mutagenesis kit. Our group recently published a modified protocol that allows the kit to be used for large-scale insertions and deletions in protein constructs. The protocol also allows for substitutions to be made at a greater efficiency than via the manufacturer's instructions.
Central to the success of modified protocol is a new primer design strategy. Read Liu et al (2008) for further details. While the strategy is straightforward, it can be time consuming to implement: especially if one requires many constructs to be designed for downstream high-throughput applications. RubyPrimer makes this task easier. ###How to Use The only input required is the coding DNA for your protein (pasted in the correct frame - click the "Translate" button to check) and an experiment string to represent the mutation, insertion or deletion you are designing. Multiple experiment strings can be submitted: separate each string with a comma (,). All inputs are case-insensitive. If your modification is close to the N or C terminals of your protein, you might want to include some flanking DNA from your backbone plasmid to prevent an error.
###Experiment Strings ####General Considerations When designing your strings, the string must be unambigious. For instance in the deletion string FTGY-DERT the sequences FTGY and DERT must only appear once in your template. 4 or 5 residues will usually suffice, but pay particular attention if your modofications occur close to poly-histidine tags. To prevent an error, always include the entire poly-His tag in your experiment string. ####Deletions Deleted regions of proteins are represented by a hyphen (-). The experiment string VNAFS-TGYA will delete all residues between the S and T. ####Insertions Insertions strings are represented by a plus sign (+). Thus the experiment string VNAFS+AAAAAA+HYTF will insert 6 alanines in between the serine and the histidine. RubyPrimer will automatically generate a codon-optimised DNA sequence to cater to your insertion. ####Substitutions Substitution strings are represented by asterisks (*). The experiment string VNAFS*C*GTHY will substitute whatever residue follows the serine with a cysteine. RubyPrimer will automatically select the best (ie lowest mismatched) codon to perform the mutation. Note that RubyPrimer can only currently substitute one amino acid per experiment. This will likely change in the future. ###Technical RubyPrimer is written in the Ruby programming language and deployed on the web via Sinatra. RubyPrimer is open-source, free software (via an MIT licence). The source code can be freely viewed at GitHub. Contrubutions are welcome.