DNA-based typing of HLA alleles occasionally results in the inability to assign a specific allele because of ambiguity in associating two or more polymorphisms to the same or to alternate homologs (cis/trans ambiguity). Since most individuals are heterozygous at a given HLA locus, the highest level of confidence in definition is obtained when the alleles are tested in isolation. By using single-strand conformation polymorphisms (SSCP) to separate heterozygous HLA-DRB1 alleles, followed by sequencing of separated conformers, we have achieved resolution of previously ambiguous assignments without the need for additional probes or primers.

Major histocompatibility complex (MHC) class I-related chain A gene (MICA) sequence-based genotyping (SBT) was attempted on a peripheral blood sample collected from a patient evaluated for hematopoietic stem cell retransplant. The electropherogram pattern of MICA SBT indicated the possibility of carrying more than two MICA alleles. Subsequent cloning and sequencing of the polymerase chain reaction products revealed the presence of three distinct MICA alleles: MICA*008:01/:04 (A5.1), MICA*007:01(A4), and MICA*002:01 (A9) in the genotype of this patient. The origin of the third extra MICA allele could not be determined and would require MICA genotyping information from other family members, which is unavailable.

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