Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 53 Materials and methods After routine PCR amplification of Y-chromosome-specific, sequence-tagged site (STS) markers as described in (5), samples were placed directly into the QIAxcel system and analyzed using the QIAxcel DNA High Resolution Kit and the QX Alignment Marker 15 bp/3 kb. The analysis included the QX DNA Size Marker 50–800 bp and the QX DNA Size Marker FX174/HaeIII. Analysis was performed with the OM500 method at 5 kV voltage and a 500 second separation time. The alignment marker was injected at 4 kV for 20 seconds and samples at 5 kV for 10 seconds. Results Results of separation of specific amplicons from the AZF regions of the Y chromosome are presented in Figure 1. In lane 3 all fragments were present: sY147 (100 bp), sY152 (125 bp), sY127 (274 bp), sY84 (326 bp), sY254-DAZ (350 bp), ZFY/ZFX (495 bp) and RBM1 (800 bp). Using the QIAxcel DNA High Resolution Kit, 12 samples can be analyzed in less than 10 minutes. Results can be displayed as a gel-like image as well as an electropherogram. The analysis of PCR products was simplified by the sharp banding pattern achieved with the QIAxcel system. The automated data acquisition enhanced analysis reliability and eliminated error in sample identification. No manipulation of the PCR samples was required before analysis on the QIAxcel system, saving time and minimizing human error. Since the QIAxcel capillary electrophoresis system uses only minute amounts of DNA through electrokinetic injection, the samples are retained for downstream procedures, such as sequencing. Figure 1. Identification of Y chromosome microdeletions. PCR was prepared with primers for detecting Y chromosome microdeletions and analyzed directly on the QIAxcel system using the QIAxcel DNA High Resolution Kit. 1: male exhibiting deletions sY152, sY147, and sY254-DAZ; 2: unknown sample exhibiting no deletions; 3: male control; 4: female control; M1: QX DNA Size Marker 50–800 bp; M2: QX DNA Size Marker FX174/HaeIII. 1 2 3 4 M1 M2 – 3000 – 800 – 600 – 400 – 300 – 250 – 200 – 150 – 100 – 50 – 15 700 – 500 – 300 – 1234 M1 M2

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