Combining bioinformatics and conventional PCR optimization strategy for one-time design of high-specificity primers for WRKY gene family using unigene database.

Gene families, like the conserved transcription factor families, evolve through gene duplications and share moderate similarity between member genes. Lack of genomic data makes it difficult to design high-specificity primers to the target genes. Furthermore, many primers under-perform in highly sensitive assays like quantitative PCR due to issues of thermodynamic nature, thereby increasing the cost and time for analysis. A methodology involving intra-species and inter-generic bioinformatic sequence comparison combined with thermodynamic estimation of primer performance was used for one-time design of gene specific primers for different WRKYs, Mitogen Activated Protein-kinases and N-methyltransferases of Coffea canephora without the aid of genome sequence resources. Out of a total 37 primer sets including 31 pairs of primers for WRKY from 34 mined WRKY Unigenes/ESTs and six pairs for genes coding for MAP kinases and NBS-LRR proteins, 32 sets exhibited high specificity of amplification upon genome analysis as well as in the high-resolution melt analysis. Furthermore, PCR optimization strategies-both in silico and experimental-indicated a superior performance of the primer sets for different applications like quantitative PCR and rapid amplification of cDNA ends. Only one set of primer resulted in mis-priming upon confirmation by DNA sequencing of the cloned amplicons. The intra-species differences and inter-generic similarities ensure high specificity of primers in all cases studied. The procedure allowed design of primers for the use in different downstream applications with high performance, specificity, yield and ease-of-use.