Table 1.
Compilation of recent investigations employing single- and multiplex PCR for the detection of YCMD.
Table 2.
Primers used for the two different multiplex setups.
Figure 1.
Y-chromosomal locations of the primers used for the two multiplex setups.
Schematic representation of the Y-chromosome similar to Simoni et al. [10] showing the positions of the multiplex primers for the detection of AZFa, AZFb and AZFc deletions. The models from Vogt et al. [3] and Repping et al. [52] are shown on the left and right, respectively. The locations of the STS primers used for the two multiplex real-time setups in this work are given in red together with their genomic positions based on the GRCh37/hg19 assembly (Feb. 2009).
Figure 2.
Testing three different DNA-binding fluorescent dyes for multiplex performance.
EvaGreen (blue), SybrGreen (green) and Syto-13 (red) were tested for peak amplitude and peak separability using the AMELxy multiplex setup. Highest performance in respect to these two criteria was obtained with the EvaGreen dye, as can be seen by the 3–20 times higher melting peaks of the individual amplicons within the multiplex reaction compared to classical SybrGreen. NTC: baseline fluorescence of EvaGreen without DNA template.
Figure 3.
The SRY multiplex setup was tested on genomic DNA from patients without deletions (PC), AZFa deletion (AZFa), AZFb+c deletion (AZFbc), AZFc deletion (AZFc) and on a non-template control (NC). Black curves represent the original melt curve (scaled on left ordinate), while the red curves represent the first derivative melt curve (scaled on right ordinate). Asterisks denote missing peaks in comparison to the positive control. The abscissas reflect temperature [°C], ordinates reflect relative fluorescence [AU]. Bottom right: Microelectrophoretic separation (Bioanalyzer) of the amplicons obtained with the different setups as above (M = size marker).
Figure 4.
The AMELxy multiplex setup was tested on genomic DNA from patients without deletions (PC), AZFa deletion (AZFa), AZFb+c deletion (AZFbc), AZFc deletion (AZFc) and on a non-template control (NC). Black curves represent the original melt curve (scaled on left ordinate), while the red curves represent the first derivative melt curve (scaled on right ordinate). Asterisks denote missing peaks in comparison to the positive control. The abscissas reflect temperature [°C], ordinates reflect relative fluorescence [AU]. Bottom right: Microelectrophoretic separation (Bioanalyzer) of the amplicons obtained with the different setups as above (M = size marker).