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Denaturing gradient gel electrophoresis: a novel method for determining Rh phenotype from genomic DNA
Author(s) -
Steers Fiona,
Wallace Maura,
Johnson Philip,
Carritt Ben,
Daniels Geoff
Publication year - 1996
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1046/j.1365-2141.1996.d01-1808.x
Subject(s) - exon , genomic dna , microbiology and biotechnology , biology , genetics , allele , rh blood group system , temperature gradient gel electrophoresis , phenotype , gel electrophoresis , dna sequencing , dna , gene , antibody , 16s ribosomal rna
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 e s antigens.