There are several programs available for designing primers for site-directed mutagenesis. Most of these programs are used to calculate the annealing temperature and to predict secondary structures. They cannot be used to design a restriction site. SiteFind is designed specifically for this.
In an easy-to-use, graphical interface, the user is prompted to enter the desired template nucleotide sequence. Then, the translated amino acid sequence is given and the user is able to select which amino acids to mutate. After that, the user can specify which restriction sites to search for, and even add additional sites if so desired. Finally, after a few seconds, a list of potential restriction sites is given. For each site, only the location closest to the desired point mutation and involving the fewest number of mutations is given. This substantially reduces the amount of information the user has to process prior to selecting the optimal sequence for site-directed mutagenesis, saving both time and money. Furthermore, SiteFind can be used for any type of mutagenesis and places no limits on the number of point mutations in the mutant sequence.
As the sequence length increases, when simply generating every possible nucleotide sequence for a given amino acid sequence and then searching for the presence of a restriction site, the time required for the search increases exponentially. If done in this manner, searches of longer than 15 bp quickly become infeasible. Our "moving window" algorithm is a novel way to drastically reduce the time required for a search, and yet does so without missing any potential sites. Because SiteFind implements this algorithm, it can process sequences up to 400 bp.
Shankarappa et. al. have published a computer program called SILMUT . SILMUT is a simple command-line program that can search a short amino acid sequence for the 30 most common, 6 bp restriction sites. It does this by translating each restriction site in all three frames and compares every possible translation with the user-specified amino acid sequence. During preparation of this manuscript, we discovered another web-based program that performs a function similar to SiteFind, called the Primer Generator . However, the Primer Generator requires the user to manually type in both the wild-type sequence and desired mutant amino acid sequence and to manually pick from hundreds of output sequences. Furthermore, it is not suitable for nucleotide sequences longer than 15 bp.
In contrast, SiteFind, automatically translates the input nucleotide sequence and allows the user to graphically select which residues to mutate. Furthermore, our window algorithm enables SiteFind to quickly and efficiently work with sequences of any length. For each restriction site, if multiple locations are found, SiteFind only gives the location closest to the desired point mutation: this means much less information for the user to parse in order to choose the best restriction site and sequence. Although not specifically designed for it, SiteFind could be used to make translational fusions between two different coding sequences. The user can specify that SiteFind give every location found for each restriction enzyme, and then run a search on a portion of both sequences. Then, through manual comparison, the user could select a restriction site found within both sequences and design the appropriate primers for introducing the necessary mutations.