SEQUENCE-ANALYSIS OF THE 2ND INTRON REVEALED COMMON SEQUENCE MOTIFS PROVIDING THE MEANS FOR A UNIQUE SEQUENCING BASED TYPING PROTOCOL OF THE HLA-A LOCUS

We here present a sequencing strategy for the HLA-A locus which is gen erally applicable for all HLA class I genes. The typing strategy is ba sed on a group-specific amplification according to the serologically d efined antigens. The PCR products carry the typing-relevant polymorphi c regions of the 2nd and 3rd exon including the 2nd intron. The sequen cing primers were designed to match conserved sequence motifs in the 2 nd intron allowing a nested sequencing approach in 3' and 5' direction . These conserved regions were identified after sequence compilation o f the 2nd intron of 143 clinical samples and 48 cell lines mostly from the 9th and 10th IHWC representing all serologically defined groups o f alleles. This strategy allowed the use of only one 5' and one 3' seq uencing primer regardless of the amplified allele. Therefore, it was p ossible to use dye terminator as well as dye primer sequencing chemist ry. The amplification strategy allowed the separation of the haplotype s in almost all cases. Thus, an assignment of heterozygous positions r equiring high sequencing quality was not necessary, allowing the appli cation of Sequenase as well as TaqPolymerase as sequencing enzyme. Con cerning the resolution of heterozygosity it is obvious that this appro ach is superior to a typing system using a single pair of generic prim ers followed by direct sequencing, since the latter technique is not c apable of defining the cis/trans linkage of polymorphic sequences and, hence, cannot exclude the presence of unknown alleles.