ENRICHMENT OF OLIGO(DG)CENTER-DOT-OLIGO(DC)-CONTAINING FRAGMENTS FROMHUMAN GENOMIC DNA BY MG2-DEPENDENT TRIPLEX AFFINITY CAPTURE()

Oligo(dG).oligo(dC)- or short poly(dG).poly(dC)- containing fragments were enriched and cloned by means of Mg2+-dependent tripler affinity c apture and subsequent cloning procedures. A library constructed after three cycles of enrichment showed that similar to 80% of the clones in the supercoiled form formed a complex with labeled oligonucleotide (d G)(34). However, while the rest of the clones retained the ability to form a complex (type I clones), 90.9% failed to form a complex when th ey were linearized. This group of DNA was abundant in the genomic DNA, although it showed only similar to 3-fold enrichment by one cycle of affinity capture, This group was further classified into two species ( types II and III) based on complex formation ability after phenol extr action. Type II clones retained the complex formation ability after tr eatment, while the human telomere [(TTAGGG)(n)] and telomere-like [(TG GAA)(n)] or [(TGGAG)(n)] sequences belonging to type III clones did no t. Serial deletion experiments and the binding assays using oligonucle otides confirmed that the repetitive units containing T(G)(n)T (n= 3-5 ) tracts or (G)(n)-motifs (n greater than or equal to 3) were the site s of complex formation for type II and III clones. On the other hand, type I clones contained poly(dG).poly(dC) tracts at least 10 nt long, and DNase I-footprinting analysis indicated that these tracts were the sites of complex formation.