[Genotyping of blood-groups by denaturant gradient gel electrophoresis (DGGE)]

The denaturant gradient gel electrophoresis (DGGE) method was used in order to simultaneously estimate the genotypes of different factors in a gel plate consisting of one sheet. A genotype analysis of the blood groups (MN, Duffy, Kidd type) and serotype (Gc system) was carried out. DNA samples extracted from the leucocytes of volunteers, whose blood group had already been proven, were used. The PCR amplification fragments were amplified from the gene which controlled each blood group. The primers were designed in order to analyze genotypes with from 1-3 base substitutions in the amplification product. The concentration of the denaturant most suitable for the DGGE method to detect each genotype of the blood groups (MN, Duffy, Kidd type and Gc system) was calculated using a computer simulation method. The denaturant concentration limit of the gel which was suitable for performing a DGGE analysis was determined to range from 15-36%. Electrophoresis was performed at 60 degrees C and with 100 V using this gel on each amplification fragment for 3 hours. The gel after electrophoresis was treated in ethidium bromide in order to detect the DNA band. The genotype (M/M, M/N and N/N) of the MN blood group, the genotype (Fya/Fya, Fya/Fyb and Fyb/Fyb) of the Duffy blood group, the genotype (Jka/Jka, Jka/Jkb and Jkb/Jkb) of the Kidd blood group and each allele (GC*1S, GC*1F and GC*2) of the Gc system were all simultaneously distinguished in one plate. As a result, it was possible to estimate multiple gene polymorphism, while substituting bases 1-3, by PCR, DGGE and ethidium bromide staining. The polymorphic results obtained using this DGGE method is very useful for cases in which a multiple gene analysis is required such as for the personal identification.