Two-step high resolution sequence-based HLA-DRB typing of exon 2 DNA with taxonomy-based sequence analysis allele assignment.

A two-step high resolution sequence-based DRB typing method was developed. The system needs only one polymerase chain reaction (PCR) to type all functional DRB alleles of a given individual. It uses a pair of generic PCR primers to amplify exon 2 DNA of all functional DRB genes and a first-step taxonomy-based sequence analysis (FSTBSA) method to assign allele groups after sequencing the PCR products with a generic primer. In the second step, group-specific primers are used to sequence the same PCR products and a taxonomy-based sequence analysis (TBSA) is used to assign alleles. Thus, both low and high resolution DRB typing can be done with PCR amplified exon 2 DNA from a single PCR reaction. Correct allele group assignment by FSTBSA was confirmed by sequencing the PCR products with group-specific primers and correctly assigned all 158 DNA samples including 34 samples pre-typed by PCR-sequence-specific primer or PCR-sequence-specific oligonucleotide probe. FSTBSA correctly assigned 116 heterozygous combinations of 81 DRB1-DRB3/4/5 haplotypes. Sixty-seven DRB1, 6 DRB3, 1 DRB4, and 3 DRB5 alleles were identified in this study. TBSA successfully resolved all heterozygous allele combinations including 31 heterozygous combinations of 33 alleles of DRB1*03, 08, 11, 12, 13, and 14 allele groups, and six heterozygous combinations of six DRB3 alleles.