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Construction of a Human Genomic Library of Clones Containing Poly(dG-dA)•Poly(dT-dC) Tracts by Mg2+-dependent Triplex Affinity Capture

1995; Elsevier BV; Volume: 270; Issue: 16 Linguagem: Inglês

10.1074/jbc.270.16.9258

1083-351X

Naoko Nishikawa, Michio Oishi, Ryoiti Kiyama,

Molecular Biology Techniques and Applications

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 Mg 2+ , Mn 2+ , or Zn 2+ revealed that the procedure worked best in the presence of Mg 2+ . Affinity enrichment was performed with Alu I-digested chromosomal DNA mixed with biotinylated (dG-dA) 17 in the presence of Mg 2+ . 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.