Ex-Ex Primer: An experimentally validated tool for designing oligonucleotides spanning spliced nucleic acid regions from multiple species.

A comparative study of existing junction-primer-designing software revealed many limitations among them. Hence, we developed a new computational program, Ex-Ex Primer, which offers many improved, user-friendly features, and reliably creates junction primers and probes. This online suite can also be used to design primers/probes from other sites of nucleic acid recombination, insertion, deletion, or splicing, and regular probes/primers. The threshold for Tm difference between the complete junctional primer vs its partial sequence, which maps to one of the junctional regions, was changed based on an important observation made during the initial experimental validations. The tool is now thoroughly checked with RT-PCR and RT-qPCR experiments with more than 250 primer pairs over a few years. The junction-primer-designing features of the software are also better than other equivalent tools. Visualizing the exons and introns across transcripts, and enabling primer designing based on information from Ensembl, are some of the unique features of this tool. The primers suggested by the tool can be used to detect the expression of known transcripts, to test the existence of predicted DNA or RNA joints via hybridization-based techniques, or for validation and in silico analysis of RNA-Seq. URL: http://resource2.ibab.ac.in/exprimer/.