title: A method to discriminate between closely related bovine major histocompatibility complex class I alleles by combining established PCR-SSP assays with RFLPs.
abstract: We have developed a polymerase chain reaction-sequence-specific primers-restriction fragment length polymorphism (PCR-SSP-RFLP) method to rapidly differentiate between the A18 and A18 variant (v) BoLA haplotypes and between A14 and A15/A15v BoLA haplotypes in Holstein/Friesian cattle. We used published SSP to PCR amplify BoLA alleles expressed in animals of known haplotype and exposed the amplicons to the restriction enzyme PvuII that was predicted to cut at a unique site in the middle of BoLA-6*01302 (A18v) and BoLA-1*00901 (A15) but not in BoLA-6*01301 (A18) or BoLA-1*02301 (A14) alleles. Whereas the method does not discriminate between the A15 and A15v haplotypes, as the BoLA-1*00902 allele associated with A15v also contains a PvuII site, we are interested in cattle of A18 and A14 haplotype for vaccine related studies. Our results also indicated that the BoLA-6*01302 (A18v) allele is much more abundant than BoLA-6*01301 (A18) in the cattle that we sampled.
authors: International Livestock Research Institute (ILRI), Vaccine Biosciences, Nairobi 00100, Kenya.
authors: N
authors: N
authors: Svitek
authors: B
authors: B
authors: Nzau
authors: L
authors: L
authors: Steinaa
authors: V
authors: V
authors: Nene
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers / Q000138:chemical synthesis / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D005091:Exons; D005787:Gene Frequency; D006239:Haplotypes; D015395:Histocompatibility Antigens Class I / Q000145:classification / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D015395:Histocompatibility Antigens Class I; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: BoLA class I molecule; PCR‐SSP‐RFLP based typing; allele abundance; haplotype