URPD: a specific product primer design tool

Background Polymerase chain reaction (PCR) plays an important role in molecular biology. Primer design fundamentally determines its results. Here, we present a currently available software that is not located in analyzing large sequence but used for a rather straight-forward way of visualizing the primer design process for infrequent users. Findings URPD (yoUR Primer Design), a web-based specific product primer design tool, combines the NCBI Reference Sequences (RefSeq), UCSC In-Silico PCR, memetic algorithm (MA) and genetic algorithm (GA) primer design methods to obtain specific primer sets. A friendly user interface is accomplished by built-in parameter settings. The incorporated smooth pipeline operations effectively guide both occasional and advanced users. URPD contains an automated process, which produces feasible primer pairs that satisfy the specific needs of the experimental design with practical PCR amplifications. Visual virtual gel electrophoresis and in silico PCR provide a simulated PCR environment. The comparison of Practical gel electrophoresis comparison to virtual gel electrophoresis facilitates and verifies the PCR experiment. Wet-laboratory validation proved that the system provides feasible primers. Conclusions URPD is a user-friendly tool that provides specific primer design results. The pipeline design path makes it easy to operate for beginners. URPD also provides a high throughput primer design function. Moreover, the advanced parameter settings assist sophisticated researchers in performing experiential PCR. Several novel functions, such as a nucleotide accession number template sequence input, local and global specificity estimation, primer pair redesign, user-interactive sequence scale selection, and virtual and practical PCR gel electrophoresis discrepancies have been developed and integrated into URPD. The URPD program is implemented in JAVA and freely available at http://bio.kuas.edu.tw/urpd/.


Introduction
URPD (yoUR Primer Design), a web-based primer design tool, combines the NCBI Reference Sequences (RefSeq), UCSC In-Silico PCR, memetic algorithm (MA) and genetic algorithm (GA) primer design methods to obtain specific primer sets. A friendly user interface is accomplished by built-in parameters setting. The incorporated pipeline operations effectively guide both, advanced and occasional users. URPD contains an automated process which produces feasible primer pairs that satisfy the specific needs of the experimental design with practical PCR amplifications. Visual virtual gel electrophoresis and in-silico PCR provide a simulated PCR environment. A comparison of practical gel electrophoresis with virtual gel electrophoresis is used to validate the PCR experiment. Wet-laboratory validation proved that the system provides feasible primers. URPD is freely available at http://bio.kuas.edu.tw/urpd/.
URPD designs specific primer sets in three steps. The first step is the template sequence input; followed by the parameter settings. In a third step, feasible primer pairs are output. Figure 1 illustrates the three steps, respectively. The following sections describes the further processes of the pipeline.

Template Sequence Input
Four input types are available in URPD: 2.1. Nucleotide Accession# key in; 2.2.
Template sequence input; 2.3. Primer pair information input; and 2.4. Coping and pasting template sequences for high throughput. These steps are illustrated below.

Nucleotide Accession# key in
The NCBI Reference Sequences (RefSeq) provides a non-redundant collection, which includes sequences from plasmids, organelles, viruses, archaea, bacteria, and eukaryotes. It contains richly annotated DNA, RNA, and protein sequences from diverse taxa. URPD, in combination with NCBI Reference Sequences (RefSeq), provides a comprehensive, standard template sequence to design primers. All Nucleotide Accession# available in RefSeq are also available in URPD. Figure 2 shows the interface for Nucleotide Accession# input.

Template sequence input
A template sequence with FASTA format or plain format can be used to enter the template sequence. It is a universal function that exists in many primer design tools and is suitable for a small-scale experiment. Figure 3 shows the template sequence paste input interface.

Primer pair information input
Many primers designed by other primer design tools are inadequately annealed to irrelevant positions. UCSC In-Silico PCR effectively searches a sequence database for a pair of PCR primers to confirm their specificity. Furthermore, an unknown template sequence can be retrieved by a primer pair and different primer pairs can be designed. Figure 4 shows the primer pair information input interface, and Figure 5 shows the parameter descriptions of UCSC In-Silico PCR, which can be opened by clicking on the "Parameters Description" hyperlink.
Paste a template sequence for primer design here

Copy/paste template sequences for high throughput
Many template sequences with FASTA format or plain format can be pasted into the template sequences paste input mask. This is suitable for a large-scale experiment with high throughput. Figure 6 shows the template sequences input interface.

Parameter Settings
After a template sequence has been imported into URPD, one of two specific primer design methods can be selected, i.e., a memetic algorithm (MA) or a genetic algorithm (GA) (Figure 7). The MA method is the recommended method for specific primer design in the system as it has been proven better than the GA method. URPD provides a sequence range selection that allows the template sequence to be trimmed (see 3.1. Sequence range selection to trim the template sequence). Primer design constraints can be individually set to allow for a more flexible experiential PCR experiment; the primer design algorithm parameters can also be adjusted to improve the primers qualities (see 3.

Select primer design method methods
When the image is pressed and the designed sequence in blue color is selected in Figure 7 as the start site, the trimmed sequence before the start site is displayed in gray color (Figure 8). By pressing the image and then clicking on the designed sequence in blue color in Figure 7 as the end site, the trimmed sequence after the end site is displayed in gray color (Figure 8). Users can select the start and the end sites via the above operations to preserve the desired sequence for their primer design. Figure 8 shows the result of the sequence range selection.

Setting of primer design constraints and primer design algorithm parameters
Advanced user can individually set the primer design constraints or primer design algorithm parameters by clicking the image or in Figure 8. Figure 9 shows the interface for the primer design constraints, and Figure 10 shows the interface for the primer design algorithm parameters. Primer design constraints in URPD are the primer length, the primer length difference, the Tm formula, the Tm (melting temperature), the Tm difference, the molar sodium (Na + ) concentration, the oligonucleotide concentration, the GC proportion, the PCR product length, the annealing number for a dimer (cross-dimer & self-dimer), the annealing number for hairpins, the mismatches allowed for specificity, and a manual region selection. Primer design algorithm parameters in URPD include the maximum generation size, the population size, the crossover probability, the mutation probability, the run time, and the number of results.

Feasible Primer Pairs
URPD provides an ordered primer pair output. The primer sets are ranked according to both their fitness value estimated by MA/GA primer design method and the melting temperature difference between designed primer pairs. The best primer pairs are always shown first. The primer pair information is comprised of the forward and reverse primers, blast specific estimation, primer position (from-to), the GC number, the GC%, Visualization). Figure 11 shows result for a URPD primer design.

Blast specific estimation
NCBI blast is used to further estimate the specificity of the designed primers via a genomic sequence database. By clicking on the icon in Figure 11, URPD performs a blast search to confirm the specificity of the primer. Figure 12 shows blast results for a designed primer.

Secondary structures
The secondary structures are marked by clear symbols. Cross-dimers are marked by the symbol ; self-dimers are marked by the symbol; hairpins are marked by , GC-clamps are marked by , and the specificity is indicated by the symbol.
The information is shown after a designed primer. Figure 11 shows that all designed primers are GC-clamps and specific. The symbols facilitate the evaluation of the primer quality for a user.

Visualization
URPD visually depicts the relationship between a designed primer set and a template sequence. By clicking on the symbol, URPD opens a page that visualizes the results for a primer pair. Figure 13 shows eliminated sequences indicated by a gray bar. Template sequence are indicated by a blue bar, the designed primer pair is indicated by a green bar, and the target sequence is indicated by an orange bar. The designed primer pair information is listed on the left of the visualization. A virtual PCR gel electrophoresis is also generated by URPD and practical PCR gel electrophoresis can be loaded into URPD (Figure 14). The virtual PCR gel electrophoresis shows the product size of the designed primer pair. This allows a user to intuitively gauge the results of the PCR experiment. At the same time, users can load their practical PCR gel electrophoresis results and compare them with the virtual PCR gel electrophoresis results for further validation of the PCR experiment.

Result file output
URPD provides an output of the results through in a text format file. In Figure 11, users can click on the "Export Results" button to perform this action. The output file contains sequence information, primer design constraints, algorithm parameters, and primer pair information, all of which are shown in Figure 15 to Figure 18, respectively.