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).