Figure 1

Overall scheme for construction of AtRTPrimer (A) and flowchart for the decision for primer specificity (B). The AtRTPrimer was created through the following three steps: homogeneity, specificity, and construction steps. In homogeneity step, we generated the candidate primers from each of simple repeats-masked cDNA sequences using Dust program and ePrimer3, and then temporarily recoded. They have similar properties such as length, melting temperature and GC content. In the specificity step, we checked whether each primer of each cDNA is specific against 28,592 cDNAs. Then, we temporarily recoded specific primers of each cDNA. Right panel (B) shows how to make decision on the specificity of a primer of a cDNA. For that decision, first we ignored exact match for a primer of a cDNA to its similar cDNAs. This exception was reconsidered in construction step. Second, we checked whether or not there are mismatches in last three bases at the end of 3'. Third, we checked whether the binding affinity between a primer and a blast hit is greater than the free energy threshold. We decided that a primer had no specificity if one of all blast hits of the primer does not satisfy these three conditions sequentially. In the construction step, we filtered out the unqualified primer pairs from all possible primer pairs generated by specific primers of each cDNA. After filtering, we added the information on the possible amplicons from a genomic DNA or a similar cDNA to the remaining primer pairs (see Additional file 1 and Additional file 2)