DENATURING GRADIENT GEL-ELECTROPHORESIS - A RAPID METHOD FOR DIFFERENTIATING BOLA-DRB3 ALLELES

The products of the BoLA-DRB3 locus are important molecules in the bov ine immune response. Several techniques have been used to study and de fine this locus but they are generally time consuming and limited in t heir ability to detect novel alleles. In this study we used denaturing gradient gel electrophoresis (DGGE), and direct sequencing, for BoLA- DRB3-typing. First, modified locus-specific primers were used in polym erase chain reaction (PCR) to amplify a 240 bp fragment of exon 2 of B oLA-DRB3 from the genomic DNA of 22 cattle and one pair of twin calves . The reverse primer included a GC-rich clamp to improve the physical separation of the BoLA-DRB3 alleles by DGGE. The denaturing gradient n eeded to produce separation of alleles was determined using perpendicu lar DGGE, and this gradient was then applied to parallel denaturing ge ls. The optimal time for producing allele separation was determined us ing a time-series analysis. The bands representing individual BoLA-DRB 3 alleles were excised from the gels, reamplified, and the nucleotide sequence determined using fluorescent-based automated cycle sequencing . The nucleotide sequences of the separated bands were then compared t o published BoLA-DRB3 alleles. A gradient of 10-15% acrylamide combine d with a 15-50% urea-formamide gradient was successfully used to separ ate BoLA-DRB3 alleles in all individuals examined. Nucleotide sequenci ng showed that the 24 animals possessed 13 BoLA-DRB3 alleles, all of w hich have been previously described. The BoLA-DRB3 genotypes included 20 heterozygotes and two homozygotes. Three BoLA-DRB3 alleles were see n in each of the twin calves, possibly due to leukochimerism. The tech nique is reliable and rapid, and avoids cloning alleles prior to nucle otide sequencing and therefore offers distinct advantages over previou s techniques for BoLA-DRB3-typing.