Comparison of oligonucleotide selection by ROCKET and Primerize. (A) Forward and reverse oligonucleotides selected by ROCKET (top) and Primerize (bottom) for T. kodakarensis tRNATrp with the annealing sites in color. (B) Density plot of ΔGtotal values for 46 sets of oligonucleotides for T. kodakarensis tRNA genes, selected by ROCKET (blue) and Primerize (green). (C) PCR cycle-dependent amounts of extended DNA obtained by DNA polymerase reactions in the presence of 1 µM oligonucleotides and ∼10 nM Dream-Taq DNA polymerase (0.01 units) were quantified. The data were fitted to a single exponential curve. Errors, SD (n = 3) are shown. (D) Time-dependent amounts of extended DNA obtained by DNA polymerase reactions in the presence of 1 µM oligonucleotides and ∼10 nM Dream-Taq DNA polymerase (0.01 units) were quantified. Errors, SD (n = 3) are shown. (E–G) Transcription yields of (E) T. kodakarensis tRNAHis, (F) Thermoplasma acidophilum tRNALeu, and (G) Thermus thermophilus tRNAPro from one-pot reactions using Primerize-selected oligonucleotides and ROCKET-selected oligonucleotides were compared. Transcription yields were quantified by 10% denaturing PAGE (7 M urea) as described in the Materials and Methods. Errors (x) from three biological replicates, SD, and P-values are shown.
