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Volume 270, Number 16, Issue of April 21, pp. 9258-9264, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Construction of a Human Genomic Library of Clones Containing Poly(dG-dA)Poly(dT-dC) Tracts by Mg-dependent Triplex Affinity Capture
DNA POLYMORPHISM ASSOCIATED WITH THE TRACTS

Naoko Nishikawa , Michio Oishi , Ryoiti Kiyama

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-dependent triplex DNA formation. Examination of triplex DNA formation in the presence of various metal ions Mg, Mn, or Znrevealed that the procedure worked best in the presence of Mg. Affinity enrichment was performed with AluI-digested chromosomal DNA mixed with biotinylated (dG-dA)in the presence of Mg. 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)repeat 30-84 base pairs in length, while some contained variants such as (dC-dT)-(dC)-(dC-dT). 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.



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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.