A new HLA-DRB1 genotyping method using single nucleotide polymorphism (SNP) analysis with multiplex primer extension reactions and its application to mixed samples.

We have improved on conventional methods for HLA-DRB1 genotyping and devised a new method that is simple, cost-effective, and adequately applicable to routine forensic practice. This method consists of group-specific polymerase chain reaction (PCR) of the exon 2 region of the HLA-DRB1 gene and simultaneous detection of single nucleotide polymorphisms (SNPs) at multiple sites using multiplex primer extension reactions. With this method, we successfully detected HLA-DRB1 genotypes from the following materials: the peripheral blood of 142 donors, 6 aged saliva stains of known DRB1 genotype stored for 5-10 years at room temperature, 10 aged bloodstains of unknown DRB1 genotype stored for 29 years at room temperature, and minimal bloodstains and saliva stains from 3 donors of known DRB1 genotypes. Furthermore, we were able to type DRB1 alleles of the minor component in mixed samples at a proportion of 1/1,000 or 1/10,000. In a criminal case, DRB1 alleles detected from mixed bloodstains on a sword found at the scene enabled us to explain the case. This method is expected to be useful for forensic medicine.