Fetal sex was also confirmed by visualization of the external genitalia after the delivery. The first commercial kit used for Y-STR amplification was the Powerplex Y23 System kit (Promega). Its reaction was performed according to the manufacturer’s instructions in a GeneAmp 9700 PCR System (Life Technologies), except by the use of 60 PCR cycles. The second commercial kit used for Y-STR amplification was the AmpFlST Yfiler PCR amplification kit (Life Technologies). Its reaction was performed according to the manufacturer’s instructions in a GeneAmp 9700 PCR System (Life Technologies), except by the use of 60 PCR cycles. The third (Mini-1) and fourth (Mini-2) multiplex reactions used for Y-STR amplification were previously

described by Asamura et al. [19], they included only mini Y-STR. The mini-1 Y-STR multiplex reaction find more (4-plex) consisted of 1.0 μL of primer Baf-A1 mix (see below), 12.5 μL Maxima Probe qPCR master mix (Fermentas) and 10 μL of extracted DNA in a 25 μL volume adjusted with DNase/RNase-free water (Fermentas). The primer concentration were as follow: DYS522 (6FAM) 0.5 μM, DYS508 (VIC) 0.6 μM, DYS632 (NED) 0.6 μM, DYS556 (PET) 1.4 μM. The PCR cycling conditions were: preincubation for 10 min

at 95 °C, 50 cycles of 15 s at 95 °C, 30 s at 60 °C and a final extension of 20 min at 60 °C. The Mini-2 Y-STR multiplex reaction (3-plex) was identical to the mini-1, except the primer mix composition DYS570 (6FAM) 0.5 μM, DYS576 (VIC) 0.5 μM, DYS540 (PET) 1.4 μM and the PCR cycling condition (preincubation for 10 min at 95 °C, 50 cycles

of 15 s at 95 °C, 30 s at 55 °C and a final extension of 20 min) at 60 °C). TC-3000 thermocycler (Techne) was used to perform both reactions. The primers for Mini-1 and Mini-2 loci were synthesized by life technologies. The Powerplex Y23 and mini-1/-2 systems were used to genotype the father’s reference sample. The reactions were performed as described above, except the number of PCR cycles that were reduced to 30 in all instances. Moreover, a total of 0.5–1.0 ng of DNA (contained in a 1.2 mm FTA punch) was used per PCR reactions. When necessary, the AmpFlSTR NGM PCR amplification kit was used to perform the kinship analysis and the reactions were performed according Tau-protein kinase manufacturer’s instructions. The PCR products were separated and detected with a 3500 Genetic Analyzer. For Yfiler, NGM and mini-1/-2 reactions, 1 μL of the amplified sample was added to 8.5 μL Hi-Di Formamide and 0.5 μL of GeneScan 600 LIZ. The electrophoresis condition was 15 s injection time, 1.2 kV injection voltage, 15 kV run voltage, 60 °C, 20 min run time, Dye Set G5 (6FAM, VIC, NED, PET and LIZ). For Powerplex Y23 reaction, 1 μL of the amplified sample was added to 10 μL Hi-Di Formamide and 1 μL of CC5 ILS Y23 (Promega). The electrophoresis condition was identical as described for Yfiler, except for the Dye Set G5 (FL, JOE, TMR-ET, CXR-ET and CC5 from Promega).