NEW MAMU DRB ALLELES USING DGGE & DIRECT SEQUENCING: MHC: AUTOIMMUNE

我们描述了一种快速但明确的方法来区分DRB 使用聚合酶链反应， 变性梯度凝胶电泳（DGGE）和直接测序。 Mamu-DRB等位基因第二外显子高度变异， 用通用的DRB引物，用DGGE分离等位基因。 DNA被 然后从从凝胶中取出的塞子中再扩增， 使用基于荧光的测序确定。 有效期 通过鉴定所有DRB等位基因证实了分型程序 对于之前通过克隆和测序表征的三只猕猴 技术. 结果我们的分析揭示了DRB等位基因， 在三只参考动物中鉴定。 利用这项技术，我们 在15只不相关的猕猴中发现了40个等位基因。 的基础上 系统进化树分析表明，14个新的DRB等位基因分别属于10个 不同的MHC-DRB谱系。 两个新的DRB 6谱系 以前在原猴和马尾猕猴中发现过。 尽管传统的DRB分型方法提供的信息有限， 我们的新技术提供了一种简单而相对快速的方法， 鉴定用于组织分型的DRB等位基因，确定个体 疾病关联和易感性鉴定和研究。 目的：鉴定猕猴主要组织相容性 复杂的（MHC）等位基因，以充分了解模型的自身免疫和 传染病 未来的发展方向这项新技术也应该 有助于正在进行的研究MHC的功能和演变，在许多 不同种类的灵长类动物 关键词恒河猴， DRB 1，键入

We described a rapid but unambiguous way to distinguish DRB alleles in the rhesus macaque using the polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The highly variable second exon of Mamu-DRB alleles was amplified using generic DRB primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and alleles were determined using fluorescent-based sequencing. Validity of this typing procedure was confirmed by identification of all DRB alleles for three macaques previously characterized by cloning and sequencing techniques. RESULTS Our analysis revealed DRB alleles not previously identified in the three reference animals. Using this technique, we identified 40 alleles in fifteen unrelated macaques. On the basis of phylogenetic tree analyses, 14 new DRB alleles were assigned to 10 different MHC-DRB lineages. Two of the new DRB6 lineages had previously been identified in prosimians and pigtailed macaques. Whereas traditional DRB typing methods provide limited information, our new technique provides a simple and relatively rapid way of identifying DRB alleles for tissue typing, determining individual identification and studies of disease association and susceptibility. OBJECTIVE: To identify rhesus macaque major histocompatibility complex (MHC) alleles for fully understanding models of autoimmune and infectious disease. FUTURE DIRECTIONS This new technique should also contribute to ongoing studies of MHC function and evolution in many different species of nonhuman primates. KEY WORDS Rhesus macaque, DRB1, typing