10) The main loci affected by increasing annealing temperature

10). The main loci affected by increasing annealing temperature

were amelogenin, D1S1656, D8S1179, D10S1248, D12S391, D16S539, D22S1045 and SE33, all of these loci dropping out at 64 °C. Decreasing annealing temperature did not have a significant effect on peak height. As annealing temperature decreased with the PowerPlex® ESX Fast Systems, the known artefact peak at 63–65 bases in yellow [16] and [17] gradually increased in intensity but never saturated. In the PowerPlex® ESI Fast Systems, there was no significant increase in intensity of any of the known artefact peaks [14] and [15], although at 56 °C a low intensity artefact peak was seen at 183–184 bases within the vWA locus. This was not present at 58 °C or at the recommended 60 °C annealing temperature. Blood

and buccal FTA® card punches generated full profiles at the recommended Volasertib datasheet 60 °C annealing temperature with all four systems. The effect of annealing temperature on loci with direct amplification PF-02341066 solubility dmso samples correlates with that observed with purified DNA. Full profiles were obtained for both blood and buccal FTA® card punches with all four fast systems at 60 °C, 58 °C and 56 °C. There was no significant increase in peak height of known artefacts at 58 °C and 56 °C. Peak height and balance with 500 pg DNA was comparable on the GeneAmp® PCR System 9700, and 96-well (0.2 mL) Veriti® thermal cyclers (Fig. 3 for 17 plexes; data not shown for 16 plexes) with similar sensitivity at 50 pg (data not shown). On the GeneAmp® PCR System 2720 thermal cycler there was a drop in signal

at TH01 (63–69% of signal on 9700) and D2S1338 (50–55% of signal on 9700) for both the PowerPlex® ESI Fast and ESX Fast Systems. This effect was overcome by raising the denaturation temperature during cycling from 96 °C to 98 °C (Supplemental Fig. 11). No additional artefacts were observed in template and Doxacurium chloride no-template amplification reactions performed on the GeneAmp® PCR System 2720 and 96-well (0.2 mL) Veriti® thermal cyclers over those noted previously on the GeneAmp® PCR System 9700 thermal cycler [14], [15], [16] and [17]. At a constant mass of DNA the overall signal doubles as the reaction volume is reduced from 25 μL to 12.5 μL. However, if the concentration is kept constant, then the overall peak heights remain consistent (See Supplemental Fig. 12 for both 17 plexes. Data not shown for 16 plexes). No new artefacts were seen in the reduced volume reactions, either in the presence or absence of DNA template (data not shown). For all four fast systems, full profiles were obtained from all replicate amplifications from each of the three donors at both full and half reactions with either a single 1.2 mm punch from a blood stain on FTA® or a blood stain on ProteinSaver™ 903 or 2 μL of a SwabSolution™ Extract (Supplemental Table 4).

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