Analysis of the HLA-DR gene locus by temperature gradient gel electrophoresis and its application for the rapid selection of unrelated bone marrow donors.
The human leucocyte antigen (HLA) class II compatibility of bone marrow donor and recipient is an essential prerequisite for the prevention of severe graft versus host disease and therefore for the successful outcome of bone marrow transplantation. In this study an efficient protocol was developed for the rapid analysis of the polymorphic HLA-DR gene locus, based on DNA-polymerase chain reaction (PCR) amplification of the variable exon II of the HLA class II DR genes and subsequent temperature gradient gel electrophoresis (TGGE). Computer-assisted melting map calculations were carried out to determine the melting behavior of the different HLA-DR fragments. Despite the high variability of the DR alleles on the nucleotide sequence level the calculations revealed a common melting domain structure of the different HLA-DR fragments, which was experimentally confirmed by perpendicular TGGE. On parallel TGGE, all samples were separated under the same electrophoretical conditions using a single PCR fragment without GC-clamp. TGGE was applied for the analysis of the DR alleles of numerous bone marrow receipt pairs and compared to the corresponding serological and DNA typing results. The TGGE patterns were found to be different for all samples with different HLA-DR typing results. Identical homoduplex and heteroduplex patterns occurred only in the case of complete genotypic HLA-DR identity as determined by direct sequencing of PCR products.
Uhrberg
M
M
Transplantation Immunology with Bone Marrow Donor Registry, Heinrich-Heine University, Düsseldorf, Germany.
Hinney
A
A
Enczmann
J
J
Wernet
P
P
D000483:Alleles; D001483:Base Sequence; D001853:Bone Marrow / Q000276:immunology; D016026:Bone Marrow Transplantation / Q000276:immunology*; D003198:Computer Simulation; D004247:DNA / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005091:Exons; D005802:Genes, MHC Class II; D006086:Graft vs Host Disease / Q000276:immunology*; D006684:HLA-DR Antigens / Q000235:genetics*; D059811:HLA-DRB1 Chains; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D013696:Temperature
D017931:DNA Primers; D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D007202:Indicators and Reagents; D004247:DNA