title: Two-step high resolution sequence-based HLA-DRB typing of exon 2 DNA with taxonomy-based sequence analysis allele assignment.
abstract: 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.
authors.affiliation: Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Luo
authors.forename: J
authors.initials: J
authors.lastname: Blanchard
authors.forename: K
authors.initials: K
authors.lastname: Brunham
authors.forename: Y
authors.initials: Y
authors.lastname: Pan
authors.forename: C X
authors.initials: CX
authors.lastname: Shen
authors.forename: H
authors.initials: H
authors.lastname: Lu
authors.forename: R C
authors.initials: RC
authors.lastname: Brunham
mesh_terms: D000483:Alleles; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D005091:Exons; D006684:HLA-DR Antigens / Q000235:genetics*; D059811:HLA-DRB1 Chains; D059826:HLA-DRB3 Chains; D059845:HLA-DRB4 Chains; D059847:HLA-DRB5 Chains; D006239:Haplotypes; D006801:Humans; D015810:Linkage Disequilibrium; D016133:Polymerase Chain Reaction
chemical_list: D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D059826:HLA-DRB3 Chains; D059845:HLA-DRB4 Chains; D059847:HLA-DRB5 Chains; D004247:DNA