DRB3 Microsatellite Analysis

The highly polymorphic microsatellite in the bovine DRB3 gene is a useful marker for polymorphism in DRB3 exon 2. A strong association between expressed DRB3 polymorphism and microsatellite alleles was revealed in studies by Ellegren et al, 1993 and in the 5th BoLA workshop. The results indicated a low frequency of microsatellite length mutations as the association was consistent across several breeds. The DRB3 microsatellite may be utilised in a PCR-based typing method of bovine class II alleles, but it does not distinguish all known DRB3 alleles.

However, it is noteworthy that in all but one instance, a given class IIa haplotype was associated with a single DRB3-MS allele. The result from the 5th workshop together with the results presented by Ellegren et al, 1993 indicate that the DRB3-MS is highly stable and, therefore, is a useful marker for DRB3 polymorphism.

Repeat Structure

The DRB3 microsatellite is composed of three repeat motifs, a stretch of at least 10 uninterrupted (TG)_n dinucleotides, a long but interrupted stretch of (GA)_n dinucleotides, and a few (CAGA)_n tetranucleotides. There were pronounced sequence differences between alleles and the results indicated that the evolution of this microsatellite has involved length mutations of the dinucleotide repeats as well as point mutations causing interruptions in the dinucleotide repeats.

Method

Two primers flanking the bovine DRB3 microsatellite are used:

• LA54
  5'-GAGAGTTTCACTGTGCAG
• LA53
  5'-CGCGAATTCCCGAGTGAGTGAAGTATCT

The LA53 primer corresponds to a conserved sequence in the 3' end of exon 2 in the bovine DRB3 gene (cf. Sigurdardóttir et al, 1991).

PCR is carried out on a thermal cycler using one end-labelled primer and thirty cycles of 60s at 94C, 30s at 54C and 60 s at 72C; the last cycle is followed by a prolonged extension for 5 minutes. The amplification products are run on 6% denaturing polyacrylamide (sequencing) gels. A sequencing ladder is used for determination of size differences between alleles.

Allele Sizes

Ellegren et al, 1993 defined alleles of 159-219 base pairs (shown on the figure) and six further alleles (of 157, 165, 167, 173, 175 and 191 base pairs) were defined in the 5th BoLA workshop. For the relationship of microsatellite typing to other methods, see the tables in the 1992 report and 1996 report.