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.
Radecke
Sarah
S
Institut für Klinische Transfusionsmedizin und Immungenetik Ulm. Abteilung Transfusionsmedizin, Universitätsklinikum Ulm, D-89081 Ulm, Germany.
Radecke
Frank
F
Peter
Ingrid
I
Schwarz
Klaus
K
D001710:Biotin / Q000378:metabolism; D002460:Cell Line; D018389:Codon, Nonsense; D004247:DNA; D004260:DNA Repair / Q000502:physiology*; D018390:Gene Targeting; D017930:Genes, Reporter; D015316:Genetic Therapy; D006801:Humans; D015335:Molecular Probes; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*
D018389:Codon, Nonsense; D015335:Molecular Probes; C022262:Okazaki fragments; D009838:Oligodeoxyribonucleotides; D001710:Biotin; D004247:DNA