Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 71 each reaction containing 5–20 ng of template DNA, 1x PCR buffer, 1.0 mM MgCl2 , 0.2 µM of each dNTP, 0.5 µM of each primer, and 1 U of QIAGEN Taq DNA polymerase. Amplification was performed on a GeneAmp® PCR System 9700 thermal cycler with an initial denaturation step at 94°C for 5 min, followed by 34 amplification cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 30 s. A final elongation step at 72°C was carried out for 5 min before storing the sample at 4°C for short-term storage, or at –20°C for long-term storage. CGF assay analysis by agarose gel electrophoresis Multiplex PCRs were analyzed by electrophoresis in 2.5% agarose gels containing 1 µg/ml ethidium bromide. Band sizes were estimated using a 1 kb molecular size marker. Gels were run at 10 V/cm until the Bromophenol Blue loading dye reached a distance of 8 cm from the loading well. Gels were visualized using UV light and photographed using a Syngene transilluminator. CGF assay analysis using the QIAxcel platform Multiplex PCRs were analyzed using the QIAxcel system using the AM320 method with an injection time of 20 s. The 15 bp–3000 bp alignment marker was used as internal standard marker and band sizes were determined using the QX 100 bp to 3 kb DNA size marker. Data were visualized using BioCalculator Software (version 3). Analysis of concordance Direct comparison of agarose gel electrophoresis with capillary electrophoresis was performed by analyzing the same four sets of 5-plex PCRs for 96 samples using both methods. The presence/absence of each gene was scored and global concordance was assessed by calculating the number of matches as a proportion of the total number of data points (i.e., 96 samples x 20 genes). Results and discussion The overall concordance between the QIAxcel and agarose gel electrophoresis was 97.4% (1870/1920). A large proportion of all mismatches (39/50) was in three of the 20 genes assayed (Figure 1); the concordance in the remaining genes was 99.3%. Every single mismatch could be traced to problems with the agarose gel data. Figure 1. Percent concordance of band calls from agarose vs. QIAxcel data (n=96). A total of 50 mismatches were observed for a global concordance of 97.4% (1870 concordant datapoints in 1920 observations, i.e., 96 samples x 20 genes). Differences were primarily found in 3 of the 20 genes of the assay, which accounted for 39 of the mismatches observed. The concordance of the remaining 17 genes was 99.3% (1621 concordant data points in 1932 observations, i.e., 96 samples x 17 genes). 85 80 75 90 95 100 G e n e 1 ( 6 1 2 b p ) G e n e 2 ( 4 8 6 b p ) G e n e 3 ( 4 0 5 b p ) G e n e 4 ( 2 9 6 b p ) G e n e 5 ( 1 9 8 b p ) G e n e 1 ( 6 1 3 b p ) G e n e 2 ( 4 4 1 b p ) G e n e 3 ( 3 0 7 b p ) G e n e 4 ( 2 8 2 b p ) G e n e 5 ( 1 7 5 b p ) G e n e 1 ( 6 3 0 b p ) G e n e 2 ( 4 8 6 b p ) G e n e 3 ( 4 0 6 b p ) G e n e 4 ( 3 6 9 b p ) G e n e 5 ( 3 0 0 b p ) G e n e 1 ( 5 5 8 b p ) G e n e 2 ( 4 6 2 b p ) G e n e 3 ( 3 9 9 b p ) G e n e 4 ( 3 0 7 b p ) G e n e 5 ( 1 8 8 b p ) False positive False negatives Misclassified Multiplex 1 Multiplex 2 Multiplex 3 Multiplex 4 Concordance (%)

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