Subgrouping of DR4 alleles by DNA heteroduplex analysis.
Amplified DNA molecules from six DR4 alleles at the DRB1 locus were denatured and cross-hybridized pairwise. Several of the DNA heteroduplexes thus generated were found to possess distinct mobilities in polyacrylamide gel electrophoresis. The degree of retardation as compared to homoduplexes depends strongly on the position of mismatched nucleotide pairs. In critical positions, the type of mispairing also influences the number of heteroduplex bands since a transversion-type substitution yields two reciprocal pairings (purine-purine or pyrimidine-pyrimidine) whereas a transition-type substitution generates symmetrical (purine-pyrimidine) pairings. Based on their heteroduplex pattern the DR4 alleles can be subdivided in four groups: group I = DRB*0401, group II = 02 and 06, group III = 03 and 04, and group IV = 05. Each group can be recognized by the heteroduplex bands generated by cross-hybridizing with group II reference DNA (either the 02 or 06). This subgrouping is obtained with a single electrophoretic run and without the use of probes. However, the alleles within groups II and III, and notably the alleles 03 and 04, which are both present in the Caucasoid population, can be distinguished only by oligonucleotide hybridization (dot blot) analysis. With this limitation, the method can be recommended either in conjunction with dot blot typing or independently, thus avoiding completely the use of probes in the cases where it is not essential to discriminate between 03 and 04. The data also show that distinguishable heteroduplexes may be generated by a single mismatch. This opens the possibility of applying the same technique to genetic systems of lower degree of polymorphism.
Sorrentino
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Dipartimento di Biologia Cellulare e dello Sviluppo, Università La Sapienza, Roma, Italy.
Cascino
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D000483:Alleles; D001483:Base Sequence; D002460:Cell Line; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D005784:Gene Amplification; D015804:HLA-DR4 Antigen / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics*
D015804:HLA-DR4 Antigen; D009692:Nucleic Acid Heteroduplexes; D004247:DNA