Enrichment of human DNAs that flank poly(dA).poly(dT) tracts by triplex DNA formation

Human DNA fragments which contain poly(dA).poly(dT) tracts and their immediate flanking regions were enriched by means of triplex DNA formation. Human DNA fragments were mixed with biotinylated (dT)34 in the presence of Mg2+ and the triplex DNA [(dT)34.poly(dA).poly(dT)] was adsorbed onto streptavidin-coated magnetic beads and the DNA fragments which formed triplexes were eluted from the beads with a buffer containing EDTA. A control experiment using a plasmid with a poly(dA).poly(dT) tract indicated that DNA fragments with the tract could be enriched over 60-fold after one cycle of the treatment. After PCR amplification, the sample was subjected to the next cycle of the affinity enrichment. After four cycles, we obtained a human genomic DNA library of clones with inserts ranging from 500 to 1000 bp among which 86% had at least one poly(dA).poly(dT) tract. No less than 60% of the clones were close or distant members of the Alu family. Sequence determination of 25 clones revealed that the length of the poly(dA).poly(dT) tracts was 14 to 37 (average 28) bp and that they were located close to either end of the fragments. While 15 clones were identified as Alu family homologues and one as a human L1 family member, nine clones were of unknown origin. None of these nine clones were a highly repeated sequence nor a part of transcriptionally active regions.