Tozaki T, Mashima S, Hirota K, Miura N, Choi-Miura N H, Tomita M
Department of Physiological Chemistry, School of Pharmaceutical Sciences, Showa University, Shinagawa, Tokyo, Japan.
DNA Res. 2001 Feb 28;8(1):33-45. doi: 10.1093/dnares/8.1.33.
We performed efficient cloning and genotyping methods for isolation of a large number of polymorphic microsatellites. The methods contain the time-efficient cloning method of constructing microsatellite-enriched libraries and the economic genotyping method of fluorescent labeling of PCR products. Eighty novel equine microsatellites cloned were efficiently isolated from the enrichment library and analyzed for genotype polymorphism. Of these, 72 microsatellites were analyzed with a good resolution. The average heterozygosity of all loci was 0.52, and the number of alleles ranged from one to 9 with an average of 4.5 alleles. The other eight loci showed multiple bands of PCR products, suggesting the occurrence of microsatellites in a repetitive element, in which the number of microsatellite repeats varies among different members of the repetitive element. We found five homologous groups at flanking regions in comparison with the flanking regions of microsatellites from DNA databases. One of them showed homology to equine repetitive element-2. In the other four homologous groups, the two groups were named equine microsatellite-linked repetitive element-1 (eMLRE-1) and equine microsatellite-linked repetitive element-2 (eMLRE-2) as novel equine repetitive elements identified from equine genome. These data should help the analysis of equine DNA sequences and the design of equine genome markers.
我们采用了高效的克隆和基因分型方法来分离大量多态性微卫星。这些方法包括构建微卫星富集文库的省时克隆方法以及对PCR产物进行荧光标记的经济基因分型方法。从富集文库中高效分离出80个新克隆的马微卫星，并对其进行基因型多态性分析。其中，72个微卫星得到了良好分辨率的分析。所有位点的平均杂合度为0.52，等位基因数从1到9不等，平均为4.5个等位基因。另外8个位点显示出PCR产物的多条带，表明微卫星存在于一个重复元件中，其中微卫星重复的数量在重复元件的不同成员之间有所变化。与DNA数据库中微卫星的侧翼区域相比，我们在侧翼区域发现了五个同源组。其中一个与马重复元件-2具有同源性。在另外四个同源组中，有两个组被命名为马微卫星连锁重复元件-1（eMLRE-1）和马微卫星连锁重复元件-2（eMLRE-2），作为从马基因组中鉴定出的新型马重复元件。这些数据应有助于马DNA序列的分析和马基因组标记的设计。
