Comparison of primers in samples spiked with human genomic DNA (gDNA).

<p>(a-c) unmodified DNA primers and (d-f) 5′-<i>o</i>-TINA modified primers. Each ○ on the efficiency curves represents one threshold cycle (Cq) determination on an amplification curve with a corresponding melting curve, reported as the first derivative. Cq determinations highlighted in red would normally have been excluded based on the amplification curve and melting curve evaluation. (a, d) Unspiked samples. (b, e) All samples and negative controls spiked with 10 ng gDNA. (c, f) All samples and negative controls spiked with 100 ng gDNA. A uniform primer concentration of 200 nM was used in all samples and negative controls. The annealing temperatures for unmodified and <i>o-</i>TINA primers were 60.0°C and 66.0°C, respectively.</p

Efficiency curves for unmodified and 5′-<i>o</i>-TINA modified primers.

<p>All experiments were conducted at an annealing temperature (T<i>a</i>) of 66.0°C and primer concentrations (C<i>primers</i>) of unmodified and <i>o</i>-TINA modified primers were compared on the same plate. The melting curves corresponding to the amplification curves used for efficiency curve determination are included for the lowest C<i>primers</i> that allows an efficiency of 100% for unmodified and 5′-<i>o</i>-TINA modified primers.</p

<i>Ortho</i>-TINA (<i>o</i>-TINA) modification attached to the 5′-end of the primer sequence.

<p><i>Ortho</i>-TINA (<i>o</i>-TINA) modification attached to the 5′-end of the primer sequence.</p

Comparison of unmodified and 5′-<i>o</i>-TINA modified primer concentrations on a temperature gradient.

<p>(a) Unmodified DNA primer concentrations from 50 nM (⧫) to 800 nM (▾). (b) 5′-<i>o</i>-TINA modified primer concentrations from 50 nM (<b>◊</b>) to 800 nM (∇). Each threshold cycle (Cq) determination is presented as mean +/− standard deviation (SD) established by triplicate measurements with 1000 copies per well of target. Inter-plate normalization was based on triplicate measurements using 800 nM of unmodified primers and an annealing temperature of 61.0°C (mean Cq for normalization was 25.11 with a SD of 0.1 on each plate).</p