HLA-B TYPING BY ALLELE SEPARATION FOLLOWED BY DIRECT SEQUENCING

Due to the enormous allelic diversity of the HLA-B locus, it has been difficult to design an unambiguous molecular typing method for the all eles at this locus. Here we describe a technique for the direct sequen cing of HLA-B alleles. Initially, HLA-B alleles were PCR-amplified aft er locus-specific reverse transcription of RNA. Alleles were then sepa rated using denaturing gradient gel electrophoresis (DGGE), which sepa rates 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 mad e. This approach was theoretically sufficient to type 92 of the 118 kn own HLA-B alleles. The majority of the remaining 26 alleles con tain d ifferences at the beginning of exon 2, a region outside the DGGE-separ ated PCR products. Therefore, we used heterozygous sequencing of this region to identify 19 of these 26 alleles, raising the resolution powe r to 111 alleles. Using this technique, we analyzed immortalized cell lines and blood Samples from several different sources. Nine immortali zed cell lines were obtained from the 10th International Histocompatib ility Workshop (IHWS) and nine were derived from aboriginal peoples. A dditionally, 25 blood samples were acquired from a panel of donors pre viously shown to be difficult to type using serological techniques. Al together, using this new method of allele separation by DGGE followed by direct sequencing, we typed 52 different alleles from 57 individual s, covering 40 serological specificities.