Multiplex PCR for STR Markers

Primers and conditions for the PCR amplification of STR markers D6S2854 and D6S2859 have previously been described (Doxiadis et al. 2011 (link); Wiseman et al. 2007 (link)). Briefly, PCR reactions were multiplexed in a 25-μl reaction volume containing 1 unit of Taq polymerase (Invitrogen, Paisley, Scotland) with 0.3 μM of the forward and reverse primer of D6S2859, 0.1 μM of the forward and reverse primer of D6S2854, 5 mM MgCl₂, 0.2 mM of each dNTP, 1× PCR buffer II (Invitrogen, Paisley, Scotland), and 100 ng DNA. The cycling parameters were a 5-min 94 °C initial denaturation step, followed by 5 cycles of 1 min at 94 °C, 45 s at 58 °C and 45 s at 72 °C. The programme was followed by 25 cycles of 45 s at 94 °C, 30 s at 58 °C and 45 s at 72 °C. A final extension step was performed at 72 °C for 30 min. The amplified DNA was prepared for genotyping and analysed on an ABI 3130XL genetic analyser (Applied Biosystems). STR analysis was performed using the Genemapper software (Applied Biosystems).