Initially, HLA-B alleles were PCR-amplified after locus-specific reverse transcription of RNA. Alleles were then separated using denaturing gradient gel electrophoresis (DGGE), which separates DNA fragments based on their sequence composition. Amplification products were excised from the gel and eluted DNA was reamplified and directly sequenced. The derived sequences were aligned to a database of published HLA-B sequences, and an initial allele assignment was made. This approach was theoretically sufficient to type 92 of the 118 known HLA-B alleles. The majority of the remaining 26 alleles contain differences at the beginning of exon 2, a region outside the DGGE-separated PCR products. Therefore, we used heterozygous sequencing of this region to identify 19 of these 26 alleles, raising the resolution power to 111 alleles. Using this technique, we analyzed immortalized cell lines and blood sample from several different sources. Nine immortalized cell lines were obtained from the 10th International Histocompatibility Workshop (IHWS) and nine were derived from aboriginal peoples. Additionally, 25 blood samples were acquired from a panel of donors previously shown to be difficult to type using serological techniques. RESULTS Altogether, using this new method of allele separation by DGGE followed by direct sequencing, we typed 52 different alleles from 57 individuals, covering 40 serological specificities. OBJECTIVE: To design an unambiguous molecular typing method for the alleles at the HLA-B locus using direct sequencing. FUTURE DIRECTIONS This will allow us to determine HLA-B locus alleles in the setting of clinical transplantation. KEY WORDS cDNA, DGGE, HLA-B, PCR, sequencing, typing
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