Nishikawa N, Oishi M, Kiyama R
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
J Biol Chem. 1995 Apr 21;270(16):9258-64. doi: 10.1074/jbc.270.16.9258.
Microsatellite DNA is a useful tool for detecting DNA polymorphisms among species or individuals, especially those among closely related individuals. We constructed a library of clones that contained poly(dG-dA).poly(dT-dC) tracts from human genomic DNA by Mg(2+)-dependent triplex DNA formation. Examination of triplex DNA formation in the presence of various metal ions Mg2+, Mn2+, or Zn2+ revealed that the procedure worked best in the presence of Mg2+. Affinity enrichment was performed with AluI-digested chromosomal DNA mixed with biotinylated (dG-dA)17 in the presence of Mg2+. A library constructed after three cycles of affinity enrichment showed that over 80% of the clones contained at least one poly(dG-dA).poly(dT-dC) tract. Most of them contained a perfect (dG-dA)n repeat 30-84 base pairs in length, while some contained variants such as (dC-dT)10-(dC)-(dC-dT)9. Using the clones from the library as a probe, we detected DNA polymorphisms associated with the repeat length of the tracts in the Japanese population. We also detected a microsatellite instability among the tracts in a cancer tissue sample.
微卫星DNA是检测物种间或个体间DNA多态性的有用工具，尤其是亲缘关系较近的个体之间。我们通过依赖Mg(2+)的三链DNA形成构建了一个包含来自人类基因组DNA的聚(dG-dA).聚(dT-dC)片段的克隆文库。在存在各种金属离子Mg2+、Mn2+或Zn2+的情况下对三链DNA形成的检测表明，该过程在Mg2+存在时效果最佳。在Mg2+存在的情况下，用与生物素化的(dG-dA)17混合的AluI消化的染色体DNA进行亲和富集。经过三轮亲和富集后构建的文库显示，超过80%的克隆包含至少一个聚(dG-dA).聚(dT-dC)片段。其中大多数包含长度为30-84个碱基对的完美(dG-dA)n重复序列，而有些包含变体，如(dC-dT)10-(dC)-(dC-dT)9。使用文库中的克隆作为探针，我们检测了日本人群中与片段重复长度相关的DNA多态性。我们还在癌组织样本的片段中检测到微卫星不稳定性。
