An advantage of using PrimerSNP is that the generated specific primers are not only allele-specific, but also genome-specific. To ensure genome-specificity, PrimerSNP takes into consideration the partial matches of primers with non-target genomic sequences. The partial sequences of a primer pair, especially sequences close to the 3' end, are critical to primer specificity [2,12]. They may have multiple matches with the target and reference genomes. To eliminate this possibility, PrimerSNP checks the first eight (default value, can be user-defined) nucleotides (starting from 3' to 5' end) to ensure that only one perfect match exists between the partial 3' end sequence and the target genomic sequences, and no match with the non-target genomic sequences.
PrimerSNP uses a binary exponent system to accurately quantify the number and position of SNPs in each primer. The weight score takes into consideration both the number and the locations of SNPs. Thus, weight score is a means to compare the specificity among the primer pairs. No other programs have attempted to quantify the specificity of primers. Our initial thought was that higher weight score could result in more specific primers. However, our experiment showed that both the weight score and the free energy of melting (ΔG°) influenced the specificity of primers. A weight score of 20,000 is critical for specificity. Below this value, 25% of primers are non-specific. If the weight score is greater than 20,000, the lower the ΔG° value, the more specific of the primers. When ΔG° value is less than -55.0, 89.9% primers are specific; when ΔG° value is less than -57.0, all primers are specific. Based on this result, we suggest the following selection criteria for the user: weight score > = 20,000 and ΔG° < = -55.0.
Since the purpose of PrimerSNP is to select reliable specific primers for detection purpose, it uses strict algorithm to filter out those primers that have partial matches with the non-target genomic sequences. Therefore, no specific primers are generated for most query sequences. Only 1% to 10% of query sequences have specific primers designed. The strictness of this program ensures a higher prediction rate (>80%).
PrimerSNP is ideal for bacterial species. It may not be suitable for virus species whose genomic sequences have high mutation rate and therefore many strains. The detection of a particular strain is usually out of the research interest.
PrimerSNP finds its potential usage in screening for risky species and disease epidemics study. The Gram-negative, xylem-colonizing bacteriumXylella fastidiosa(Xf) is the causative agent of several important diseases including Pierce's disease (PD) of grape vines, almond leaf scorch disease (ALSD) and citrus variegated chlorosis (CVC) . Citrus variegated chlorois (CVC), caused by the 9a5c strain, is not known to occur in America. Should Xf-CVC be introduced into California, preventing its spread and establishment in citrus growing areas would be very difficult and perhaps impossible. The availability of multiple specific primers could be used to reliably identify this pathogen from thousands of environmental samples. Similarly, specific primer design could be used in identifying pathogens of citrus canker disease, caused byXanthomonas axonopodispv. citri (Xac), whose genomic sequences are available and no occurrence has been reported in California.
Strain differentiation is another area that PrimerSNP could be used. Since each primer contains unique SNPs for the strain, the change in these SNPs could be easily detected by PCR. Instead of sequencing many target regions, standard PCRs on these strains using specific primers that are designed by PrimerSNP can provide a rapid clue to this question for biologists. Multiple specific primers can also be used for strain genotyping research.
Currently, the specific primer design using PrimerSNP for higher organisms such as fungi is greatly limited due to the availability of genomic sequences. With the rapid development of sequencing technology such as 454, the cost and time would be significantly reduced. PrimerSNP will certainly be a useful tool for higher organisms.