Snibson K J, Maddox J F, Fabb S A, Brandon M R
Centre for Animal Biotechnology, School of Veterinary Science, University of Melbourne, Parkville, Victoria, Australia.
Anim Genet. 1998 Oct;29(5):356-62. doi: 10.1046/j.1365-2052.1998.295351.x.
In the present study we characterize allelic variation of polymorphic OLA-DQA1 and OLA-DQA2 genes in sheep. To achieve this, PCR primers were designed to independently amplify the second exons of OLA-DQA1 and OLA-DQA2 genes. Single strand conformation polymorphism (SSCP) gel analyses reveals that there are at least 12 distinct OLA-DQA2 sequences, 10 of which have been characterized by sequencing. Six distinct OLA-DQA1 alleles have been sequenced in sheep and we can detect at least seven DQA1 alleles, including a null allele, by SSCP analysis. The second exon of the OLA-DQA2 gene is more polymorphic than the equivalent region of the OLA-DQA1 gene. Thirty-two per cent of nucleotide and 49% of amino acid sites showed variation at the DQA2 locus, compared to 20% of nucleotide and 33% of amino acid sites for DQA1. Phylogenetic analysis of DQA sequences from a number of species show that sheep DQA1 sequences group together and are more similar to bovine DQA1 sequences than to sheep DQA2 alleles. The majority of OLA-DQA2 sequences are on the same main branch of the phylogenetic tree as bovine DQA2 sequences. However, three sheep DQA2 sequences have a tendency to group with putative bovine DQA3 sequences rather than to other ovine DQA2 alleles. A variety of SSCP gel conditions were tried in order to develop a typing system for the OLA-DQA2 gene. We describe a set of PCR and SSCP conditions which distinguish between all known OLA-DQA2 alleles.
在本研究中，我们对绵羊中多态性OLA - DQA1和OLA - DQA2基因的等位基因变异进行了表征。为此，设计了PCR引物以独立扩增OLA - DQA1和OLA - DQA2基因的第二个外显子。单链构象多态性（SSCP）凝胶分析显示，至少有12种不同的OLA - DQA2序列，其中10种已通过测序进行了表征。已对绵羊中的6种不同的OLA - DQA1等位基因进行了测序，并且通过SSCP分析我们可以检测到至少7种DQA1等位基因，包括一个无效等位基因。OLA - DQA2基因的第二个外显子比OLA - DQA1基因的等效区域具有更高的多态性。在DQA2位点，32%的核苷酸位点和49%的氨基酸位点存在变异，而DQA1分别为20%的核苷酸位点和33%的氨基酸位点。对多个物种的DQA序列进行的系统发育分析表明，绵羊DQA1序列聚集在一起，并且与牛DQA1序列的相似性高于与绵羊DQA2等位基因的相似性。大多数OLA - DQA2序列与牛DQA2序列位于系统发育树的同一主要分支上。然而，三种绵羊DQA2序列倾向于与假定的牛DQA3序列聚类，而不是与其他绵羊DQA2等位基因聚类。为了开发OLA - DQA2基因的分型系统，尝试了多种SSCP凝胶条件。我们描述了一组能够区分所有已知OLA - DQA2等位基因的PCR和SSCP条件。
