Establishment of a sequence‐based typing system for BoLA‐DRB3 exon 2

Abstract

A rapid, high-resolution sequence-based typing (SBT) system for BoLA-DRB3 exon 2 was developed. Amplification of the entire exon was achieved by a fully nested PCR with locus-specific primers and sequencing was performed directly on the PCR product. Heterozygous sequence data were obtained by automated sequence analysis of both alleles. Forward and reverse sequence data were assembled to improve identification of all heterozygous positions. Specific software (Haplofinder, Roslin Institute Software, Roslin, UK) was designed for allele assignment. Fifty-four females from a Holstein-Charolais resource herd cross, their 12 sires and five unrelated Holstein animals were used to establish the method. In parallel, these animals were typed by DRB3 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to confirm the results. Polymerase chain reaction-RFLP analysis defined 15 known types in the 71 animals, while SBT of the same animals showed 19 known alleles. Subsequently, 72 more animals from the same resource herd were typed by the established SBT method without PCR-RFLP typing. This SBT strategy and the Haplofinder software can be applied to the analysis of any polymorphic locus for which suitable locus-specific primers and allelic sequences are available.

Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein

“…marginale based on cELISA [22]. Alleles for the DRB3 gene were determined by direct sequencing of PCR product, using published primers [23]. DNA was extracted from whole blood (Qiagen, Valencia, CA) as described above.…”

“…DNA was extracted from whole blood (Qiagen, Valencia, CA) as described above. The DRB3 alleles were amplified using PCR [23, 24] and the PCR product was sequenced (Eurofins Operon-SimpleSeq, Huntsville, AL). Allele assignment was done using Assign ATF 1.5 (Conexio Genomics, Fremantle, Australia).…”

The diversity of bovine MHC class II DRB3 and DQA1 alleles in different herds of Japanese Black and Holstein cattle in Japan

“…The number of BoLA-DRB3 alleles detected in each herd ranged from 9 to 20. By contrast, two cattle (one from JB2 and one from HO1, each carrying three DRB3 alleles as assessed by PCR-SBT and sequencing) were excluded from the study, as this could have resulted from leukochimerism, as previously reported in dizygotic twins (Miltiadou et al, 2003). The alleles with frequencies N5% were DRB3*0201, *0503, *1001, *1101, *1201, *1501 and *1601 in Japanese Black, and DRB3*0101, *1001, *1101, *1201, *1501 and *2703 in Holstein.…”

Characterization and genotyping of the DRB1 gene of the major histocompatibility complex (MHC) in the Marmota monax, animal model of hepatitis B

“…For the detection of alleles in heterozygous samples, the Aurkisek 2.0 script (Arrieta-Aguirre et al, 2006) was used, which was previously developed by our group and works on HAPLOFINDER (Miltiadou et al, 2003) that assigns alleles to diploid sequences. Thus, a database was generated containing alleles previously cloned.…”