A GENERIC SEQUENCING BASED TYPING APPROACH FOR THE IDENTIFICATION OF HLA-A DIVERSITY

Sequencing Based Typing (SET) is a generic approach for the identifica tion of HLA-A polymorphism. This approach includes the high resolution typing of the HLA-A broad reacting groups, HLA-A subtypes and will id entify new alleles directly. The SET approach described here uses a lo cus specific amplification of DNA from exon 1 to exon 5. The resulting 2,022 bp PCR product serves as a template for the subsequent sequenci ng reactions. Amplification is followed by direct sequencing of exons 2, 3 and 4 in both orientations with fluorescently labeled primers to define all polymorphic positions leading to a high resolution typing r esult. In this study the sequence of exons 2 and 3 of a panel of 49 ce ll lines was determined. In addition, the exon 4 region of 35 cell lin es was also sequenced to evaluate the exon 4 polymorphism. The HLA-A t ype of most of the cells could be identified by sequencing only exons 2 and 3. However, the sequence of exon 4 was required to discriminate A0201 from A*0209 and A*0207 from A*0215N. In this panel, an identica l new ''HLA-A0103'' was identified in two Caucasian samples. (C) Amer ican Society for Histocompatibility and Immunogenetics, 1997. Publishe d by Elsevier Science Inc.