Warner Jeffery F, Harpole Michael G, Crerar Lorelei D
Department of Biology, George Mason University, 4400 University Drive, Exploratory Hall Room 1218, MSN 3E1, Fairfax, VA, 22030, USA.
Center for Applied Proteomics and Molecular Medicine, George Mason University, 10920 George Mason Circle, Manassas, VA, 20110, USA.
Methods Mol Biol. 2017;1606:205-217. doi: 10.1007/978-1-4939-6990-6_14.
Microsatellite DNA can provide more detailed population genetic information than mitochondrial DNA which is normally used to research ancient bone. The methods detailed in this chapter can be utilized for any type of bone. However, for this example, four microsatellite loci were isolated from Steller's sea cow (Hydrodamalis gigas) using published primers for manatee and dugong microsatellites. The primers DduC05 (Broderick et al., Mol Ecol Notes 6:1275-1277, 2007), Tmakb60, TmaSC5 (Pause et al., Mol Ecol Notes 6: 1073-1076, 2007), and TmaE11 (Garcia-Rodriguez et al., Mol Ecol 12:2161-2163, 2000) all successfully amplified microsatellites from H. gigas. The DNA samples were from bone collected on Bering or St. Lawrence Islands. DNA was analyzed using primers with the fluorescent label FAM-6. Sequenced alleles were then used to indicate a difference in the number of repeats and thus a difference in individuals. This is the first time that H. gigas microsatellite loci have been isolated. These techniques for ancient bone microsatellite analysis allow an estimate of population size for a newly discovered St. Lawrence Island sea cow population.
微卫星DNA能够提供比常用于研究古代骨骼的线粒体DNA更详细的群体遗传信息。本章详述的方法可用于任何类型的骨骼。然而，在这个例子中，利用已发表的海牛和儒艮微卫星引物，从斯特勒海牛（Hydrodamalis gigas）中分离出了四个微卫星位点。引物DduC05（Broderick等人，《分子生态学笔记》6：1275 - 1277，2007年）、Tmakb60、TmaSC5（Pause等人，《分子生态学笔记》6：1073 - 1076，2007年）以及TmaE11（Garcia - Rodriguez等人，《分子生态学》12：2161 - 2163，2000年）均成功从斯特勒海牛中扩增出微卫星。DNA样本取自白令岛或圣劳伦斯岛上采集的骨骼。使用带有荧光标记FAM - 6的引物对DNA进行分析。然后利用测序的等位基因来显示重复数目的差异，进而表明个体间的差异。这是首次分离出斯特勒海牛微卫星位点。这些古代骨骼微卫星分析技术使得对新发现的圣劳伦斯岛海牛种群的种群规模进行估计成为可能。
