IDENTIFICATION AND CHARACTERIZATION OF NEW BOLA-DRB3 ALLELES BY HETERODUPLEX ANALYSIS AND DIRECT SEQUENCING

A sample of 52 mixed-breed dairy cattle (Holstein Friesian and Jersey) and 51 beef cattle (Hereford) from south-east queensland was studied. The second exon of BoLA-DRB3 was amplified by polymerase chain reacti on (PCR), and polymorphisms were detected by heteroduplex analysis. A large number of different heteroduplex patterns indicated extensive se quence polymorphism. Direct sequencing of PCR products from 17 homozyg otes and cloning and sequencing of PCR product from two heterozygotes resulted in the identification and characterization of four novel alle les. The previously described allele BoLA-DRB32A is characterized by an amino acid deletion at position 65. We have identified three animal s that are homozygous for this amino acid deletion, indicating that th e deletion is unlikely to result in loss of function. Two of these ani mals had allele BoLA-DRB32A, and one had a novel allele with codon 65 deleted but differing from BoLA-DRB32A at a number of other amino ac id positions. In conclusion, heteroduplex analysis allows rapid discri mination between homozygotes and heterozygotes, and enables rapid iden tification of new BoLA-DRB3 alleles.