Asymmetric PCR-SSCP: a Useful Tool for Detection of OLA-DRB1 (MHC Class II) Gene Polymorphism in Slovak Improved Valachian Sheep

Tkáãiková a. , M. R. , Bhide I . Mikula: Asymmetric PCR-SSCP: a Useful Tool for Detection of OLA-DRB1(MHC class II) Gene Polymorphism in Slovak Improved Valachian Sheep. Acta Vet. Brno 2005, 74: 275-278. Detection of OLA-DBR1 (exon 2) gene polymorphism is presented in the paper. Rapid and inexpensive polymorphism detection method, namely, single stranded conformation polymorphism (SSCP) was assessed. Modification of the SSCP to asymmetric PCR-SSCP enabled a more simplified assay for the clustering the individuals into distinct groups (profiles) on the basis of band patterns, as only single stranded amplicons were detected. A total of 400 Valachian sheep were included in the study. In this cohort, 25 distinct clusters were noticed. Among 25 groups the frequency of k profile was the highest (28%), followed by profile e (21%), p (16 %), w (9%) and d (9%). The homologous SSCP as well as asymmetric-PCR-SSCP patterns were observed among the twins: This finding has shown the sensitivity of both methods to segregate the individuals on the basis of their allelic forms. MHC, OLADRB1 gene, SSCP, polymorphism, sheep The major histocompatibility complex (MHC) plays a central role in the immune response of vertebrates. The extreme polymorphism in MHC genes enables the host to recognize enormous numbers of foreign peptides to trigger an immune reaction. The class II gene region of the sheep MHC (OLA) has an organization similar to that of humans (Scot t et al. 1987). Within this region, two sub-regions, namely, DR and DQ exhibit higher polymorphism (Amil ls et al. 1998). Among OLA class II genes, the DRB1 locus is highly polymorphic. In particular the polymorphism is present in exon 2, which encodes the antigen-binding site. To date nearly 160 OLA-DRB1 alleles have been recorded from various sheep breeds (Konnai et al. 2003). In the population genetics, analysis of large number of samples is a prerequisite. Though the DNA sequencing is gold standard for most of the phylogenetic studies, the cost requisite for such analysis is quite high as well as it is time consuming. Recently, simple and rapid techniques like PCR-RFLP (restriction fragment length polymorphism) and DGGE (denaturing gradient gel electrophoresis) have been used by different researchers for the detection of MHC gene polymorphism (Aldridge et al. 1998; Konnai et al. 2003). These techniques enable to group the individuals into clusters on the basis of gene polymorphism. However, the amount and cost of restriction enzymes required for analyzing large numbers of samples may jeopardize the use of RFLP. SSCP (single stranded conformation polymorphism) offers a simple and inexpensive method for genotyping. Hitherto, SSCP has been extensively used in biomedical research, especially for rapid bacterial genotyping. In this study we used simple SSCP as well as modified asymmetric PCR-SSCP for DRB1 genotyping in autochthonous Valachian sheep breed. To our knowledge, no report is as yet available elaborating the OLA-DBR1 alleles present in the Valachian breed. Considering the recent interest in Valachian sheep breeding ACTA VET. BRNO 2005, 74: 275–278 Address for correspondence: Doc. MVDr. o. Tkáãiková, PhD. Laboratory of Biomedical Microbiology and Immunology University of Veterinary Medicine Komenského 73, 041 81 Ko‰ice, Slovakia Phone: + 421 556 336 614 Fax: +421 556 323 666 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm in Middle and Eastern European countries it is necessary to know the natural disease resistance ability of this sheep breed. The results presented in this study create a benchmark for further gene polymorphism study influencing the ability of disease resistance or susceptibility of this breed. Materials and Methods Animals A total of 400 autochthonous Valachian sheep (including 20 twins) were included in the study from different farms located in Eastern Slovakia. Genomic DNA was isolated from blood leukocytes and DNA was stored at -20 °C until used. PCR amplif icat ion Two PCR primers forward (5’-TCT CTG CAG CAC ATT TCC TGG-3’) and reverse (5’-CTC GCC GCT GCA CAG TGA AAC-3’) (Ammer et al. 1992) were used to amplify entire exon 2, with flanking intron. The total product length was 279 bp. Reactions were performed by using 0.6 – 1.3 μg of genomic DNA in a 50 μl final volume. The PCR reaction mixture contained 100 μM of each dNTP, 0.5μM of each primer, 1.5 mM MgCl2, and 1U of Taq polymerase. The conditions for PCR amplification were as follows: 94 oC for 5 min, followed by 35 cycles of 94 °C for 90 s, 60 °C for 30 s and 72 °C for 90 s with final extension at 72 °C for 10 min. To produce single stranded amplimers, asymmetric PCR was performed. In short, the PCR reaction mixture was maintained as described previously except the concentration of DRB1R primer was reduced by 100 folds. For amplification of asymmetric amplimers the same PCR cycles were used as described above. Single-Strand Conformational Polymorphism Analysis Five 5 μl of each amplified product was mixed with 3 μl of loading dye (98% formamide, 10 mM EDTA, 0.025% bromophenol blue, 0.025% xylene-cyanol). Double stranded amplimers were subjected to denaturation at 95 °C for 8 min and samples were rapidly cooled on ice. Denaturated as well as single stranded amplimers derived form asymmetric PCR were loaded on acrylamide isacrylamide (37.5:1; Bio-Rad) gels. In the study we tried 6, 8, 10 and 14% polyacrylamide gels. The concentration of glycerol in gel was kept either 5% or 10% to compare the effect on resolution. Electrophoresis was performed using 200 V at 5 °C in 0.5 × TBE buffer. Gels were silver-stained according to the method of Bassam et al. (1991). Different electrophoresis runs (14, 16, 18 and 20 hours) were also tried in the study.