Supplementary Methods. Detailed description of methods used for primer design.


Fig. S1. Comparison of three different design strategies for targeting Ohtaekwangia sequences. Gel runs of PCR products before and after digestion with the restriction enzyme HinfI, which cuts near the centre of Ohtaekwangia amplicons. Lane 1 contains a 100 base pair ladder. Lanes 2 and 3 contain PCR products obtained with Ohtaekwangia primers designed with hybridization efficiency alone (strategy #1) before and after digestion respectively. The PCR products in lanes 4 and 5 were obtained with primers designed using elongation efficiency (strategy #2), and lanes 6 and 7 with primers designed using elongation efficiency and an induced mismatch in the 6th position from the 3′-end. Intensity profiles from the top to bottom of lanes 3, 5 and 7 are shown in (B), (C) and (D) respectively. The digested (shorter) target amplicon (Ohtaekwangia) is coloured in green, whereas undigested non-target amplicons are coloured in red. Note that the reverse primer's target site in strategy #3 (lanes 6 and 7) is shifted towards the forward primer by 31 nucleotides relative to the reverse primer's target site used for strategy #2 (lanes 4 and 5). This difference in amplicon sizes (Table S2) explains the shorter digested and undigested product lengths in lanes 6 and 7 relative to lanes 4 and 5 respectively.


Fig. S2. Comparison of decreased hybridization efficiency with decreased elongation efficiency. Amplification curves for the Mycobacterium template. This figure illustrates (A) decreased hybridization efficiency and (B) decreased elongation efficiency.

A. Equal initial concentrations of template were amplified with an annealing gradient from 50°C to 75°C.

B. Equal initial concentrations of template were amplified using either perfect match forward (F) and reverse (R) primers (solid line), or one primer with a 3′-terminal mismatch (primer/template, dashed lines).


Fig. S3. Flowchart of describing how to design a primer step-by-step using the online Design Primers web tool.


Table S1. Terminal mismatched primers and observed elongation efficiencies.


Table S2. Efficiency of elongation of 3′ non-terminal mismatches.


Table S3. Results obtained with primers designed to discriminate alleles of the Human IDH2 gene using qPCR (n = 3).


Table S4. Primer sets used to validate the primer design methodology.


Table S5. Average efficiency of elongation of 3′ terminal mismatches separated by the penultimate primer nucleotide.


Table S6. DNA templates and perfect match primer sequences used to determine relative elongation efficiency of 3′ terminal mismatches.

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