STRATEGY FOR ANALYSIS OF HLA-DQB1 ALLELES USING A COMBINATION OF REAL-TIME-PCR WITH FLUORESCENCE DNA-PROBES (FLUOROTYPING) AND AUTOMATED SEQUENCING.
EK Petershofen, C Seidl, C Drosten, R Frank, G Holzberger, and E Seifried. Institute of Transfusion Medicine and Immunohematology, RCBDS, Frankfurt, Germany.


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In this report we describe a new strategy to determine DQB1 allele specific DNA sequences using a combination of a real-time-PCR fluorescence assay (Taqman, Perkin Elmer, Applied Biosystems 7700 SDS) and fluorescence-based DNA sequencing (Taq cycle sequencing using dye terminators, Perkin Elmer ABD automated DNA sequencer 373A). DQB1 typing is performed from genomic DNA samples as follows: (A) pre-amplification by two separate PCR-SSP reactions (DQB1*05/*06 vs *02/*03/*04); (B) determination of PCR products by fluorotyping with a total of 11 SSP reactions (10 within exon 2, one for exon 3) and (C) automated sequence-based-typing (SBT) for DQB1*03/05/*06 subtypes. Fluorotyping and sequencing reactions were evaluated with reference DNA samples (12th IHWS/controlled laboratory DNA). The fluorotyping probe was conjugated with 5'FAM (fluorescence: em 490/ ex 520) as the reporter dye and 3' TAMRA as the quencher dye. Eight typing reactions can be performed in a 96-optical-tube plate; each PCR step takes approx. 55 minutes. This strategy offers the advantage of automated PCR amplification of the DQB1 locus at a intermediate resolution level (amplification step) and detection of positive reactions by fluorescence measurement through degradation of a DQB1-specific DNA probe (hybridisation step). High resolution typing can be easily performed by SBT.