Denaturing gradient gel electrophoresis: a novel method for determining Rh phenotype from genomic DNA
- 1 August 1996
- journal article
- Published by Wiley in British Journal of Haematology
- Vol. 94 (2) , 417-421
- https://doi.org/10.1046/j.1365-2141.1996.d01-1808.x
Abstract
Denaturing gradient gel electrophoresis (DGGE) was carried out on PCR products amplified from exons 2 and 5 of RHD and RHCE. Exon 2 of RHD and exon 2 of the C allele of RHCE have an identical sequence, which differs from that of the c allele of RHCE. One band representing D and/or C, and another representing c, could be distinguished by DGGE of exon 2 amplifications of genomic DNA from individuals with the appropriate Rh phenotype. C and c could only be distinguished in D‐negative samples. Exon 5 of RHD and exon 5 of the E and e alleles of RHCE all have different nucleotide sequences. Bands representing D, E and e could be distinguished following DGGE of the products of exon 5 amplification of genomic DNA from individuals with red cells of the appropriate Rh phenotype. In samples from individuals with VS+ red cells (V+ or V−) there was a shift of the band representing e. Sequencing demonstrated that VS is associated with a RHCE e sequence with a single base change predicting a Leu245 → Val substitution in the Rh polypeptide. This substitution may be responsible for the VS and es antigens.Keywords
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