SEQUENCING BASED TYPING OF HLA-DRB GENES PRIOR TO BONE MARROW TRANSPLANTATION: COMPLETE EXON SEQUENCING BASED ON THE CONSERVED INTRONIC DIVERSITY.
          Katja Kotsch, Jenny Wehling, Rainer Blasczyk,  Department of Hematology and Oncology, Blood Bank, Charité, Campus Virchow-Klinikum, Humboldt-University, Berlin, Germany.
          SBT is the method of choice if high resolution typing of the DRB loci is required. The sequencing method applied should be able to define the cis/trans linkage of sequence motifs. The major drawback of exon-based group-specific amplification is the inability to determine the sequence of the 5' and 3' ends of exon 2. Our strategy is based on 1st and 2nd intron motifs adjacent to exon 2 of the DRB genes which offer the possibility to establish clear serology-related amplification strategies. The method allows the complete analysis of the polymorphic exon 2 and a more reliable definition of the cis/trans linkages of sequence motifs by applying group-specific intron-located primer mixes separating the haplotypes. It delivers a higher resolution for the DR52 associated DRB1 group than the standard procedure using exon-based amplification primers since the groups DR3, DR12 and DR14 are amplified by specific primer mixes. The DR11 and some DR13 alleles do not show any individual sequence motif in intron 1 and intron 2. They have to be amplified, depending on the second haplotype, either by a multispecific DR3,11,13,14 primermix, or in case of a DR3,11 or DR11,14 heterozygosity by multispecific primer mixes for the separate amplification of DR11. In 19 primer mixes, 19 sense and 8 antisense primers are used to amplify HLA-DRB alleles corresponding to the serologically defined specificities DR1-DR14, DR51-DR53. The PCR products vary in size from 385-1035 bp. The sequence homology of the 3' end of intron 1 facilitates the application of only 3 different forward sequencing primers for all DRB alleles. Using the advantageous AmplitaqTM FS dye terminator chemistry, our sequencing strategy is superior compared to existing strategies in gaining complete exon 2 sequences and unambiguous typing results regardless of a growing HLA sequence databank.