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 available for amplification and typing of a polymorphic region in the Human Leukocyte Antigen (HLA) DQ alpha DNA sequence. Following amplification of the DQ alpha region with the PE kit, typing strips are processed. These strips contain immobilized DNA probes designed to distinguish six possible HLA DQ alpha alleles. It has been observed in this laboratory 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 suggested that a potential source of this weak-signal is the non-specific amplification of a HLA DX alpha gene sequence. To demonstrate the relationship 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 which the amplified product spans the homologous DQ alpha region encompassing the DNA probes present on the typing strips. DX alpha DNA sequences from various DQ alpha genotypes were amplified and hybridized to DQ alpha typing strips. We have demonstrated that DX alpha PCR products do not always hybridize to the 1.1 probe on the typing strips. Sequence analysis of DX alpha PCR products show that this region is polymorphic which may explain why the occurrence of the "1.1 weak-signal" is unpredictable. We have further analyzed the effect of DNA template concentration for the DQ alpha amplification protocol and have shown that regulation of PCR input DNA optimizes the amplification and typing protocols for HLA DQ alpha alleles and minimizes the potential observation of the "1.1 weak-signal."
Crouse
C A
CA
Palm Beach County Sheriff's Crime Laboratory, West Palm Beach, FL.
Vincek
V
V
Caraballo
B K
BK
D001483:Base Sequence; D005554:Forensic Medicine / Q000379:methods*; D005784:Gene Amplification; D006683:HLA-DQ Antigens / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*
D006683:HLA-DQ Antigens