DNA Sequence Assignment to the BoLA-DRB3.2*17 PCR-RFLP allele

LIRON, J.P.; DIAZ, S.; RIPOLI, M.V.; BOUZAT, J.L; PERAL-GARCIA, P; GIOVAMBATTISTA,G.

Basic and Applied Genetics

Sociedad Argetina de Genetica

Año: 2003 vol. 16 p. 19 - 20

1666-0390

The typing method based on PCR-RFLP for the BoLA-DRB3 exon 2 was extensively used in populational and disease association studies. To date, more than fifty PCR-RFLP variants were described in domestic cattle. Six BoLA-DRB3 variants correspond to alleles that have not been sequenced yet (DRB3.2*4, 14, 17, 25, 39 and 40). Genomic DNA was purified from a semen sample corresponding to an individual of the Aberdeen Angus breed. Amplifications of the second exon of BoLA-DRB3 gene were performed by heminested – PCR and digested with RsaI, BstYI and HaeIII enzymes. The PCR product was cloned and sequenced. The restriction site analysis of the reported DNA sequence (Genbank Accession number AF317664) confirmed that the sequence corresponded to the BoLA-DRB3.2*17 allele. Blast analysis showed that our sequence is 100% identical to the sequence described in Japanese breed by Takeshima et al. (2001) (Genbank accession number AB048732), but this allele was not previously defined by PCR-RFLP. The predicted aminoacid sequence from this allele corresponds to BoLA-DRB3*2502 allele. In conclusion, the DNA sequence reported here can be assigned to the allele BoLA-DRB3.2*17, which was previously defined solely by PCR-RFLP. The typing method based on PCR-RFLP for the highly polymorphic BoLA-DRB3 exon 2 was developed in 1992 (van Eijk et al, 1992). To date, up to 54 PCR-RFLP variants were described in bovines (www.projects.roslin.ac.uk/bola/bolahome.html). This strategy was used to report BoLA-DRB3 types 1 - 31(van Eijk et al., 1992), 32 - 40 (Gelhaus, et al., 1995) and 41 - 54 (Maillard, et al., (1999). The  restriction patterns of the majority of the reported PCR-RFLP types correspond to one or more BoLA-DRB3 exon 2 DNA sequences, and are based on the predicted patterns found in alleles defined in BoLA workshops. Some types were defined from sequences which are not yet confirmed as DRB3 alleles. The BoLA-DRB3.2*30 corresponded to a genotyping error. Finally, 6 out of 53 BoLA-DRB3 variants correspond to alleles that have not been sequenced yet (DRB3.2*4, 14, 17, 25, 39 and 40). Some of these alleles were only defined in population studies. In the present note we assigned a DNA sequence to the BoLA-DRB3.2*17 PCR-RFLP allele. Genomic DNA was purified from a semen sample corresponding to an individual of the Aberdeen Angus breed. Using an extraction buffer (HCl-Tris 50 mM, DTT 35 mM, EDTA 25 mM, N-laurylsarcosine 2%) with 20 ml of 10 mg/ml proteinase k, the DNA was isolated by precipitation with 10 M ammonium acetate and precipitate with isopropanol. Amplifications of the second exon of BoLA-DRB3 gene were performed by heminested – PCR with primers HL030, HL031 and HL032 (van Eijk, et al., 1992). Amplification products were digested with RsaI, BstYI and HaeIII enzymes and alleles were defined as previously described (van Eijk, et al. 1992; Gelhaus, et al. 1995; Maillard, et al., 1999).