Nested PCR allows the characterization of TaqI and PstI RFLPs in the second exon of the caprine MHC class II DRB gene.
A nested polymerase chain reaction (PCR) method has been developed to obtain a specific amplification of the second exon of the caprine MHC class II DRB gene. The specificity of this method has been verified by cloning and sequencing the PCR product and comparing its sequence to 21 previously published caprine DRB second exon allelic variants. Nucleotide identity between this sequence (Caae-DRB23) and other caprine DRB alleles ranged between 85.6% (Caae-DRB22) and 96.5% (Caae-DRB5). Caae-DRB5 and Caae-DRB23 sequences diverged in five amino acid substitutions (70, 71, 73, 74, 78), all of them placed at the antigen binding site. Likewise, the restriction polymorphism of the caprine DRB second exon has been analyzed and two different restriction patterns have been found depending on the presence or absence of a TaqI site and a PstI site at positions 122 bp and 241 bp of the PCR product respectively. TaqI and PstI RFLPs were also analyzed in other artiodactyla species. While PstI RFLP was found not only in goats but also in cattle, sheep and pigs, TaqI RFLP was only detected in goats. In all of these species close associations were detected between the presence of TaqI and PstI restriction sites and amino acid substitutions at positions 40 and 78 respectively, suggesting that PCR restriction fragment length polymorphism (RFLP) could be a useful tool in relating amino acid substitutions at critical positions with disease resistance.
Amills
M
M
Departament de Patologia i Producció Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Francino
O
O
Sánchez
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase; D015252:Deoxyribonucleases, Type II Site-Specific; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D005091:Exons / Q000235:genetics; D005784:Gene Amplification; D005802:Genes, MHC Class II / Q000235:genetics*; D006041:Goats / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D012194:RNA-Directed DNA Polymerase; D019914:Taq Polymerase
D017931:DNA Primers; D004247:DNA; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific