RH D D GENOTYPING BY QUANTITATIVE POLYMERASE CHAIN-REACTION AND CAPILLARY ZONE ELECTROPHORESIS/

A safe and reliable method for determining RhD type (positive or negat ive) and zygosity (D/D or D/d) could have applications, for instance, in the prediction of the D genotype of a father in couples where there is an RhD-negative woman at risk of fetal alloimmunization. Capillary zone electrophoresis (CZE) is proposed as a novel, reliable and power ful method for quantitative evaluation of polymerase chain reaction (P CR) products. RhD is determined by amplifying a 136 bp region common t o the RhCcEe and RhD genes and a 186 bp region specific of the RhD gen e. RhD positive and negative samples are identified by polyacrylamide gel slab electrophoresis, followed by silver staining of the DNA bands , and by CZE in sieving liquid polymers, with direct on-line peak dens itometric evaluation by exploiting the intrinsic UV absorbance of the DNA fragments at 254 nm. The CZE method allows not only a reliable ass essment of the RhD type (the presence of both 136 bp and 186 bp fragme nts indicating an RhD-positive type, the presence of only the 136 bp f ragment indicating an RhD-negative type), but also a rapid determinati on of the zygosity based on the quantitative expression ratio of the 1 36 bp/186 bp pair. Thus, a 2:1 peak ratio clearly indicates a D/D homo zygous individual, whereas a 3:1 peak ratio gives evidence of a D/d he terozygous individual.