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

Abstract

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, OLA-DRB1 gene, SSCP, polymorphism, sheepThe 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 (Scott et al. 1987). Within this region, two sub-regions, namely, DR and DQ exhibit higher polymorphism (Amills 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...