Construction of a human genomic library of clones containing poly(dG-dA).poly(dT-dC) tracts by Mg(2+)-dependent triplex affinity capture. DNA polymorphism associated with the tracts.
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.
Nishikawa
N
N
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Oishi
M
M
Kiyama
R
R
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004276:DNA, Satellite / Q000737:chemistry; D015723:Gene Library; D006801:Humans; D008274:Magnesium / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011089:Polydeoxyribonucleotides / Q000737:chemistry*; D011110:Polymorphism, Genetic
D004276:DNA, Satellite; D011089:Polydeoxyribonucleotides; C050366:poly(dG-dA)n.poly(dC-dT)n; D004247:DNA; D008274:Magnesium