Blood samples from the peripheral blood of pregnant women were collected in 2–3 ml EDTA-K2 anticoagulative tubes. Plasma separation from these blood samples was performed using a two-step centrifugation method, as previously described (22 (link)). Then, 20 µl Protease K (20 mg/ml) and 200 µl plasma were mixed, and centrifuged briefly after adding 200 µl Buffer AL (Qiagen, Dusseldorf, Germany). The solution was incubated at 56°C for 10 min, centrifuged briefly, 200 µl absolute ethyl alcohol was added, mixed, and subsequently transferred to the spin columns (Qiagen). The spin columns were centrifuged for 1 min at 6,000 × g, 500 µl Buffer AW2 (Qiagen) was added prior to centrifugation for 3 min at 20,000 × g, and then the filtrate was discarded. Next, the spin columns were centrifuged for 1 min at 20,000 × g, and the DNA was eluted with 60 µl Buffer AE (Qiagen), separated into aliquots and stored at −80°C prior to use.
The amplification of the SRY gene (Y chromosomal material) using nested PCR was performed as previously described (22 (link)). The primers used for this nested PCR were as follows: SRY-138 forward (F) (5′-TACAGGCCATGCACAGAGAG-3′) and SRY-138 reverse (R) (5′-TGTTGTCCAGTTGCACTTCG-3′) (first round); and SRY-116F (5′-GCACAGAGAGAAATACCCGAAT-3′) and SRY-116R (5′-GCACTTCGCTGCAGAGTACC-3′) (second round).
The amplification of the SRY gene (Y chromosomal material) using nested PCR was performed as previously described (22 (link)). The primers used for this nested PCR were as follows: SRY-138 forward (F) (5′-TACAGGCCATGCACAGAGAG-3′) and SRY-138 reverse (R) (5′-TGTTGTCCAGTTGCACTTCG-3′) (first round); and SRY-116F (5′-GCACAGAGAGAAATACCCGAAT-3′) and SRY-116R (5′-GCACTTCGCTGCAGAGTACC-3′) (second round).