165-P: NEW PCR AND SEQUENCING REAGENTS FOR SEQUENCE BASED TYPING OF HLA-DRB1 EXON 2 AND EXON 3

Abstract

Design PCR and sequencing primers for subsequent use in the development of an assay prototype that enables the full length sequencing of HLA-DRB1 Exons 2 and 3. AlleleSEQR® HLA-DRB1 reagents currently include primers for amplifying and sequencing Exon 2. For the present HLA-DRB1 reagents, the 5’ PCR primers are located in intron 1 and the 3’ PCR primers span the Exon 2 and intron 2 boundary. We have designed and developed a new set of HLA-DRB1 Exon 2 PCR and sequencing primers where the 5’ and the 3’ PCR primers are located in intron 1 and intron 2 respectively to amplify the entire Exon 2 followed by sequencing for complete Exon 2 coverage. We follow a similar approach where the PCR and sequencing primers are placed in the intronic regions (introns 2 and 3) to amplify and sequence the entire Exon 3. Sequencing reactions were standardized for the 3100 and the 3500xL (Life Technologies, CA) genetic analyzers. We tested 500 HLA typed DNA samples from UCLA and IHWG registries and compared the HLA typing results with our newly designed set of reagents for Exon 2 and Exon 3 to the results generated with the existing current AlleleSEQR® HLA-DRB1 set of reagents. All DNA amplified and sequenced for Exon 2 and Exon 3. All sequences were 100% concordant with the published HLA-DRB1 typing results for the 500 DNA samples obtained from UCLA and IHWG registries. Some HLA-DRB1 alleles can only be identified by specific nucleotide base calls that are located in the last few bases of Exon 2. Sequencing the entire Exon 2 could potentially resolve such ambiguities and in addition some alleles are further confirmed based on the ambiguities typed in these last few bases. The ability to sequence through Exon 3 allows the typing of additional alleles that have identical base sequences in Exon 2. Sequencing additional HLA-DRB1 gene regions upfront may avoid the need to use alternative HLA typing test methods and ambiguity resolution primers thus conserving sample DNA, reagents and labor. Wadia: Celera / Quest Diagnostics: Employee; Stockholder. Drews: Celera / Quest Diagnostics: Employee. Sigua: Celera / Quest Diagnostics: Employee. Jiang: Celera / Quest Diagnostics: Employee. Bost: Celera / Quest Diagnostics: Employee.