Cryer Nicholas C, Butler David R, Wilkinson Mike J
School of Biological Sciences, University of Reading, Reading, Berkshire, RG6 6AS, UK.
Plant Methods. 2005 Aug 18;1(1):3. doi: 10.1186/1746-4811-1-3.
Large-scale genetic profiling, mapping and genetic association studies require access to a series of well-characterised and polymorphic microsatellite markers with distinct and broad allele ranges. Selection of complementary microsatellite markers with non-overlapping allele ranges has historically proved to be a bottleneck in the development of multiplex microsatellite assays. The characterisation process for each microsatellite locus can be laborious and costly given the need for numerous, locus-specific fluorescent primers.
Here, we describe a simple and inexpensive approach to select useful microsatellite markers. The system is based on the pooling of multiple unlabelled PCR amplicons and their subsequent ligation into a standard cloning vector. A second round of amplification utilising generic labelled primers targeting the vector and unlabelled locus-specific primers targeting the microsatellite flanking region yield allelic profiles that are representative of all individuals contained within the pool. Suitability of various DNA pool sizes was then tested for this purpose. DNA template pools containing between 8 and 96 individuals were assessed for the determination of allele ranges of individual microsatellite markers across a broad population. This helped resolve the balance between using pools that are large enough to allow the detection of many alleles against the risk of including too many individuals in a pool such that rare alleles are over-diluted and so do not appear in the pooled microsatellite profile. Pools of DNA from 12 individuals allowed the reliable detection of all alleles present in the pool.
The use of generic vector-specific fluorescent primers and unlabelled locus-specific primers provides a high resolution, rapid and inexpensive approach for the selection of highly polymorphic microsatellite loci that possess non-overlapping allele ranges for use in large-scale multiplex assays.
大规模基因图谱分析、定位及基因关联研究需要一系列特征明确且具有多态性的微卫星标记，这些标记要有不同且宽泛的等位基因范围。历史证明，选择具有不重叠等位基因范围的互补微卫星标记是多重微卫星检测技术发展的一个瓶颈。鉴于需要大量位点特异性荧光引物，每个微卫星位点的特征分析过程可能既费力又昂贵。
在此，我们描述了一种简单且低成本的方法来选择有用的微卫星标记。该系统基于将多个未标记的PCR扩增产物混合，然后将它们连接到一个标准克隆载体中。第二轮扩增利用靶向载体的通用标记引物和靶向微卫星侧翼区域的未标记位点特异性引物，产生代表混合样本中所有个体的等位基因图谱。为此，我们测试了各种不同大小DNA混合样本的适用性。评估了包含8至96个个体的DNA模板混合样本，以确定广泛人群中单个微卫星标记的等位基因范围。这有助于解决在使用足够大以检测许多等位基因的混合样本与混合样本中包含过多个体从而导致稀有等位基因过度稀释以至于不在混合微卫星图谱中出现的风险之间的平衡问题。来自12个个体的DNA混合样本能够可靠地检测出混合样本中存在的所有等位基因。
使用通用载体特异性荧光引物和未标记的位点特异性引物为选择高度多态性的微卫星位点提供了一种高分辨率、快速且低成本的方法，这些位点具有不重叠的等位基因范围，可用于大规模多重检测。
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