Rh genotyping
Fresh blood samples (100 µl) were mixed with 200 µl 10mM Tris buffer of pH 7.0 containing 5 mM EDTA (TE), frozen, and stored in -20 °C. After thawing, DNA was isolated with phenol-chloroform method and resuspended in the final volume of approximately 50 µl TE. One µl of the sample was added to the 9 µl reaction mix (0.1 µl 5U Taq DNA polymerase (SBD, Tartu, Estland), 1 µl 10×Taq DNA polymerase buffer, 0.6 µl 25 mM MgCl2, 0.2 µl 10 mM dNTPs, 0.1 µl primer 5´ CCCAGTGACCCACATGC 3´, 0.1 primer 5´ CCTACATTGTGCTGCTGG 3´, and 7 µl ddH20), mixed and loaded to 96-well plate, overlaid with 10 µl mineral oil and amplified (forty cycles: 95°C/30sec-56°C/30sec-72°C/30sec). After the amplification, 0.3 µl of LCGreenPlus (Invitrogen, USA), was added to each well and the plate was analyzed on LightTyper (Roche, Switzerland), in temperatures 75-85°C. One peek at 81°C was present in the profile of Rh-negative homozygotes and two peeks at 82°C and 83.5°C in the profile of Rh heterozygotes (the ratio of heights 2 : 1) and Rh-positive homozygotes (the ratio of heights 1 : 1).