Sun H S, Kirkpatrick B W
Department of Meat and Animal Science, University of Wisconsin-Madison 53706, USA.
Mamm Genome. 1996 Feb;7(2):128-32. doi: 10.1007/s003359900032.
Dinucleotide microsatellites are useful for gene mapping projects. Depending upon definition of conservation, published estimates of dinucleotide microsatellite conservation levels vary dramatically (30% to 100%). This study focused on well-characterized genes that contain microsatellites in the human genome. The objective was to examine the feasibility of developing microsatellite markers within genes on the basis of the assumption of microsatellite conservation across distantly related species. Eight genes (Gamma-actin, carcinoembryonic antigen, apolipoprotein A-II, cardiac beta myosin heavy chain, laminin B2 chain, MHC class I CD8 alpha chain, c-reactive protein, and retinoblastoma susceptibility protein) containing large dinucleotide repeat units (N > or = 15), complete genomic structure information, and homologous gene sequences in a second species were selected. Heterologous primers were designed from conserved exon sequences flanking a microsatellite motif. PCR products from bovine and porcine genomic DNA were tested for the presence of microsatellite sequences by Southern blot hybridization with biotin-labeled (CA)12 oligonucleotides. Fragments containing microsatellites were cloned and sequenced. Homology was verified by sequence comparisons between human and corresponding bovine or porcine fragments. Four of sixteen (25%) cross-amplified PCR products contained dinucleotide repetitive sequences with repeat unit lengths of 5 to 23. Two dinucleotide repetitive sequences showed microsatellite length polymorphism, and an additional sequence displayed single-strand conformational polymorphism. Results from this study suggest that exploitation of conserved microsatellite sequences is a useful approach for developing specific genetic markers for comparative mapping purposes.
二核苷酸微卫星对于基因图谱绘制项目很有用。根据保守性的定义，已发表的二核苷酸微卫星保守水平估计值差异很大（30%至100%）。本研究聚焦于人类基因组中含有微卫星的特征明确的基因。目的是基于远缘物种间微卫星保守性的假设，研究在基因内开发微卫星标记的可行性。选择了八个基因（γ-肌动蛋白、癌胚抗原、载脂蛋白A-II、心脏β肌球蛋白重链、层粘连蛋白B2链、MHC I类CD8α链、C反应蛋白和成视网膜细胞瘤易感蛋白），这些基因含有大的二核苷酸重复单元（N≥15）、完整的基因组结构信息以及第二个物种中的同源基因序列。从微卫星基序侧翼的保守外显子序列设计异源引物。通过用生物素标记的（CA）12寡核苷酸进行Southern印迹杂交，检测来自牛和猪基因组DNA的PCR产物中微卫星序列的存在。克隆并测序含有微卫星的片段。通过人与相应牛或猪片段之间的序列比较验证同源性。16个交叉扩增的PCR产物中有4个（25%）含有二核苷酸重复序列，重复单元长度为5至23。两个二核苷酸重复序列显示微卫星长度多态性，另一个序列显示单链构象多态性。本研究结果表明，利用保守的微卫星序列是开发用于比较图谱绘制目的的特定遗传标记的有用方法。
