Physical incorporation of a single-stranded oligodeoxynucleotide during targeted repair of a human chromosomal locus

Background Targeted gene repair is an attractive method to correct point-mutated genes at their natural chromosomal sites, but it is still rather inefficient. As revealed by earlier studies, successful gene correction requires a productive interaction of the repair molecule with the target locus. The work here set out to investigate whether DNA repair, e.g., mismatch repair, or a direct incorporation of the correction molecule follows as the step upon the initial interaction. Methods Single-stranded 21mer oligodeoxynucleotides (ODNs) of sense orientation were directed towards point-mutated enhanced green fluorescence protein transgene loci in HEK-293-derived cell clones. First gene repair assays compared ODNs carrying the canonical termini 5'-phosphate and 3'-OH with their respective variants harbouring non-canonical termini (5'-OH, 3'-H). Second, a protocol was established to allow efficient recovery of integrated short biotin-labelled ODNs from the genomes of gene-corrected cells using streptavidin-coated beads in order to test directly whether transfected ODNs become bona fide parts of the target locus DNA. Results Oligodeoxynucleotides with canonical termini were about 34-fold more efficient than their counterparts carrying non-canonical termini in a phosphorothioate-modified backbone. Furthermore, biotinylated fragments were successfully recovered from genomic DNAs of gene-corrected cells. Conclusions The experiment showed that ODNs are incorporated into a mammalian genome. This unravels one early repair step and also sets an unexpected example of genome dynamics possibly relevant to other ODN-based cell techniques. Copyright (c) 2005 John Wiley & Sons, Ltd.