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Table 2 PCR program for amplification of AR, SRY and DYS14 genes Steps PCR Program for AR gene PCR Program for SRY gene PCR Program for DYS14 gene Initial denaturation 95 °C for 10 mins 95 °C for 10 mins 95 °C for 10 mins Denaturation 95 °C for 20 s 95 °C for 1 min.

The program for amplification included an initial denaturation step at 95°C for 5 min, followed by 40 cycles of 95°C for 45 s and 58°C for 30 s. Product amplification was detected using SYBR Green fluorescence during the 58°C step.

The program for amplification for the first stage was 95°C for 15 min, 30 cycles (95°C for 30 s, 62°C for 1 min, 72°C for 2 min) and 72°C for 10 min.

The standard PCR program for amplification consisted of an initial denaturation step at 94°C for 4 min, followed by 35 cycles of 30 sec at 94°C, 30 sec at the indicated annealing temperature, n min at 72°C (n = size in kilobases of amplified product), and a final elongation at 72°C for 5 min.

The thermocycler program used for amplification of pnpE1 and pnpE2 was the following: (i) initial denaturation at 95°C for 5 min; (ii) 10 cycles of denaturation at 95°C for 1 min, primer annealing at 48°C for 30 s and fragment amplification 72°C for 1 min; (iii) followed by 25 cycles of denaturation at 95°C for 1 min, primer annealing at 63°C for 15 s and fragment amplification 72°C for 1.5 min.

The program used for amplification was: 95°C for 10 minutes, followed by 40 cycles of 95°C 10 seconds, 60°C 10 seconds and 72°C 10 seconds.

The program used for amplification was: denaturation (95°C for 15 min ., 2-step amplification and quantification (92°C for 15s, 60°C for 1 min. and one fluorescence measurement), melting curve program (60 90°C with a heating rate of 0.1°C per second and one fluorescence measurement per second).

Program for DNA amplification cycle was: 95°C for 30 s for one cycle, followed by 80 cycles of amplification protocol: denaturation at 95°C for 10 s, annealing and extension at 60°C for 30 s. PCR amplification and melting curves were analyzed by MxPro software (Stratagene).

The program for PCR amplification was as follows: initial denaturation at 94°C for 5 min, 35 cycles of 94°C for 30 s, 53°C for 45 s, 72°C for 60 s, and a final extension at 72°C for 10 min.

The program for quantification amplification was 3 min at 94 °C, 20 s at 95 °C, 30 s at 59 °C and 30 s at 72 °C for 36 cycles in 25  μl reaction volume.

The PCR program for SSR amplification consisted of the following steps: 94°C for 2 min followed by 35 cycles of 94°C for 30 s, 55°C for 45 s and 72°C for 1 min, then a final step of 72°C for 5 min (modified from Tangphatsornruang et al. (2008) [ 13]).