Genotyping BoLA-DRB3 alleles in Brazilian Dairy Gir cattle (Bos indicus) by temperature-gradient gel electrophoresis (TGGE) and direct sequencing.
BoLA-DRB3 is a gene of the major histocompatibility complex (MHC) in cattle. The product of the BoLA-DRB3 gene is a beta chain of an MHC class II molecule, a glycoprotein expressed on the surface of antigen-presenting cells (APCs). Responses of CD4+ T lymphocytes to peptides are dependent on the presentation of peptide ligands bound to class II molecules on APCs. Genotyping of the BoLA-DRB3 gene is relatively complex due to the extensive polymorphism of this locus. Current techniques for assignment of genotypes are polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), direct sequencing of PCR products, cloning-sequencing, polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP), and denaturant-gradient gel electrophoresis. These techniques are time-consuming, do not discriminate all possible alleles, or are not readily reproducible. The objective of this study was to genotype BoLA-DRB3 using temperature-gradient gel electrophoresis (TGGE) to separate alleles before sequencing. PCRs using 28 DNA samples from Gir Dairy cattle (a Brazilian breed of Bos indicus) were submitted to TGGE. New PCR products were generated from separated alleles, purified, and sequenced. Allele separation was possible in 21 out of 26 heterozygote samples (81%). Results indicate that two sequence reads (forward and reverse) were sufficient for accurate genotyping of BoLA-DRB3 alleles. Separation of alleles by TGGE provides high-throughput, reliable typing of BoLA-DRB3, which is critical in disease association studies in cattle.
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Embrapa Gado de Leite, Brazilian Organization for Agricultural Research, National Dairy Cattle Research Center, Juiz de Fora, Brazil.
Martinez
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Coutinho
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D000483:Alleles; D000818:Animals; D000938:Antigen-Presenting Cells / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004586:Electrophoresis; D005091:Exons; D005838:Genotype; D006023:Glycoproteins / Q000737:chemistry; D006579:Heterozygote; D000949:Histocompatibility Antigens Class II / Q000235:genetics*; D006801:Humans; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D013696:Temperature
C423282:BoLA-DRB3 antigen; D006023:Glycoproteins; D000949:Histocompatibility Antigens Class II; D010455:Peptides; D004247:DNA