ANALYSIS AND INTERPRETATION OF THE HLA DQ-ALPHA 1.1 WEAK-SIGNAL OBSERVED DURING THE PCR-BASED TYPING METHOD

The Perkin-Elmer (PE) AmpliType DQ alpha Forensic Kit is currently ava ilable for amplification and typing of a polymorphic region in the Hum an Leukocyte Antigen (HLA) DQ alpha DNA sequence. Following amplificat ion of the DQ alpha region with the PE kit, typing strips are processe d. These strips contain immobilized DNA probes designed to distinguish six possible HLA DQ alpha alleles. It has been observed in this labor atory and others that in a single source DNA sample, it is possible to detect a weak signal on the 1.1 specific allele dot when the samples' genotype clearly does not contain the 1.1 allele. It has been suggest ed that a potential source of this weak-signal is the non-specific amp lification of a HLA DX alpha gene sequence. To demonstrate the relatio nship of the DX alpha gene to the 1.1 nonspecific signal, we designed biotinylated DX alpha PCR primers specific for a 178 bp region in whic h the amplified product spans the homologous DQ alpha region encompass ing the DNA probes present on the typing strips. DX alpha DNA sequence s from various DQ alpha genotypes were amplified and hybridized to DQ alpha typing strips. We have demonstrated that DX alpha PCR products d o not always hybridize to the 1.1 probe on the typing strips. Sequence analysis of DX alpha PCR products show that this region is polymorphi c which may explain why the occurrence of the ''1.1 weak-signal'' is u npredictable. We have further analyzed the effect of DNA template conc entration for the DQ alpha amplification protocol and have shown that regulation of PCR input DNA optimizes the amplification and typing pro tocols for HLA DQ alpha alleles and minimizes the potential observatio n of the ''1.1 weak-signal.''