Polymerase chain reaction (PCR)-based typing methods are molecular diagnostic techniques widely used in biological and biomedical studies. The level of discriminatory power of PCR-based typing depends upon the molecular marker targeted. Therefore, identifying appropriate DNA target regions for primer annealing is a crucial step because these regions must be conserved within the target taxa but must vary among related taxa [1, 2].
Recent advances in next-generation sequencing technology are enabling genome sequencing projects at a significantly lower cost, even for non-model organisms. The resulting increase in the amount of genomic data available, combined with bioinformatics tools, have led to the identification of highly informative markers, such as microsatellites and orthologous or taxa-specific genes [3].
Microsatellites or single sequence repeats (SSR) are tandem repeated stretches of short nucleotide motifs, usually ranging from 1 to 6 bp, ubiquitously distributed in the genomes of eukaryotic organisms. These regions are more prone to genetic variation and the differences in the length of individual SSR loci can be easily screened by PCR. In fact, this technique has been useful for several studies including strain typing and population genetics [4, 5]. The conventional method of SSR discovery is time consuming and costly. Therefore, in silico mining analysis has been used to improve marker identification [6, 7].
Orthologs are homologous proteins in different species that evolved from a single ancestral sequence and are related by speciation events. These sequences tend to show more functional similarity than other homologs. The identification of orthologous genes is useful in a wide range of contexts, such as inference of gene function, comparative genomics, evolutionary conservation and sequence variability [8]. Due to their importance, many tools have been developed to predict ortholog groups, including the widely used software OrthoMCL [9].
The demand is increasing for bioinformatic tools that automate analysis of genomic data generated by next-generation sequencing technology [10]. An example is the development of automated procedures to facilitate species-specific primer design for diagnostic methods [2]. Several web-based tools for facilitating primer design are available [11], but many of them are written mainly to assist in the primer design process and are not meant to search for targets and analyze primer specificity. The use of fully automated methods to search for molecular markers and the availability of genomic data for a growing number of taxa would increase the efficiency of PCR-based genotyping applications. Moreover, this strategy might save time and resources because the in silico evaluation of the candidate primers against the target genomic sequence are performed prior to testing them in the laboratory [12]. Thus, there is a need for a tool to search for appropriate genomic target regions and then design specific primers towards the selected markers.
In this context, we have developed TipMT to meet the growing demand for easy-to-use software that facilitates the design of primers that target molecular markers to distinguish the genomic sequences of different taxa. This program only requires genomic sequences of a target species and offers, as an output, specific primers for a given taxa.