Sequence-based genotyping of the sheep MHC class II DRB1 locus
Sequence-based genotyping of the sheep MHC class II DRB1 locus
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DOI：
10.1007/s00251-009-0410-y
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发表时间：
2010-01-10
期刊：
影响因子：
3.2
通讯作者：
Tassi, Riccardo
中科院分区：
文献类型：
--
作者：
Ballingall, Keith T.;Tassi, Riccardo
免疫多态性数据库（IPD）为一系列不同物种的主要组织相容性复合体（MHC）基因座上的等位基因提供了单一的命名法。纳入绵羊II类DRB 1序列的最低要求是提交包括来自至少两个独立聚合酶链反应（PCR）的第二外显子内的所有多态性位点。为了满足这些要求，我们开发了一种基于DNA的基因分型方法，用于快速分析家绵羊DRB 1位点的等位基因多样性。利用位于外显子2两侧内含子内的一系列引物和来自代表15个不同品种和杂交品种的214只绵羊的基因组DNA，获得了38个Ovar-DRB 1等位基因的完整外显子2序列。该序列资源允许开发基因座特异性引物的通用集合，其扩增包括第二外显子内的所有多态性位点的片段。PCR产物的双向序列分析提供了复合序列，其中每个多态性位点由相应的国际生物化学联合会核苷酸代码表示。在IPD或国家生物技术信息中心数据库中保存的等位基因的基本局部比对搜索工具搜索允许鉴定单个等位基因序列。低水平的纯合性（7.48%）的队列和验证以前的基因型样本证实了广泛的等位基因特异性的这种方法。它改进了目前可用的方法，并广泛适用于调查与疾病的抗性或易感性的联系的研究中的MHC多样性的分析。
The immunopolymorphism database (IPD) provides a single nomenclature for alleles at the major histocompatibility complex (MHC) loci for a range of different species. The minimum requirements for inclusion of a sheep class II DRB1 sequence is a submission that includes all polymorphic sites within the second exon from at least two independent polymerase chain reactions (PCR). In order to meet these requirements, we have developed a DNA-based genotyping method for the rapid analysis of allelic diversity at the DRB1 locus in domestic sheep, Ovis aries. Using a series of primers located within introns flanking exon 2 and genomic DNA from a cohort of 214 sheep representing 15 different breeds and crossbreeds, the complete exon 2 sequences of 38 Ovar-DRB1 alleles were obtained. This sequence resource allowed the development of a generic set of locus-specific primers which amplify a fragment that includes all polymorphic sites within the second exon. Bidirectional sequence analysis of the PCR product provides a composite sequence where each polymorphic site is represented by the corresponding International Union of Biochemistry nucleotide code. A Basic Local Alignment Search Tool search of alleles held within the IPD or National Center for Biotechnology Information databases allows individual allele sequences to be identified. Low levels of homozygosity (7.48%) within the cohort and verification of previously genotyped samples confirmed the broad allelic specificity of this method. It improves on currently available methods and is broadly applicable to the analysis of MHC diversity in studies investigating linkages with resistance or susceptibility to disease.