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

• DOI: 10.1034/j.1399-0039.2003.00080.x
• Publication Date: 2003-07-11T12:52:43Z
• Authors: D. Miltiadou; A.S. Law; G.C. Russell
• Keywords: Male; 0301 basic medicine; Heterozygote; Major histocompatibility complex; Molecular Sequence Data; Sequence-based typing; Polymerase Chain Reaction; Sensitivity and Specificity; 03 medical and health sciences; Gene Frequency; Consensus Sequence; Animals; BoLA; Polymorphism; Alleles; Sequence Deletion; Class II; Base Sequence; Agricultural Sciences; Histocompatibility Antigens Class II; Exons; Sequence Analysis, DNA; AGRICULTURAL SCIENCES; Cattle; Female; Polymorphism, Restriction Fragment Length

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