RESUMO
The introduction of a 5'-tailed duplex (5'-TD) fragment into cells corrects a base-substitution mutation in a target DNA. We previously reported that the gene correction efficiency was improved when a frameshift type of second mismatch was present â¼330 bases distant from the target position, between the target DNA and the 5'-TD fragment. In this study, the effects of the second mismatches on the gene correction were further examined. Base-base mismatches 332 bases distant from the target position slightly enhanced gene correction, but less efficiently than the previously studied frameshift mismatches. The gene correction efficiency was also increased when the distance between the target position and the second frameshift mismatch was changed to â¼270 bases. These results suggested that the introduction of an appropriate second frameshift mismatch into the 5'-TD fragment improves the gene correction efficiency.
Assuntos
Região 5'-Flanqueadora/genética , Pareamento Incorreto de Bases/fisiologia , Proteínas de Escherichia coli/genética , Terapia Genética/métodos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Proteínas Ribossômicas/genética , Sequência de Bases , Mutação da Fase de Leitura , Células HeLa , Humanos , Mutagênese Sítio-Dirigida/métodos , Proteína S9 RibossômicaRESUMO
Base sequence conversion in target DNA is achieved when a 5'-tailed duplex (TD) is introduced into cells. In this study, the effects of target DNA cleavage on sequence conversion with a TD were examined. Plasmid DNAs with and without cleavage near the target position were each introduced into HeLa cells, together with the TD. The cleavage promoted the sequence alteration efficiency by ca. 7-fold. These results suggested that the sequence conversion efficiency with the TD fragment is increased when an artificial nuclease introduces cleavage near the target site.
Assuntos
Sequência de Bases , Clivagem do DNA , DNA/metabolismo , Células HeLa , Humanos , PlasmídeosRESUMO
A 5'-tailed duplex (TD) DNA corrects a base-substitution mutation. In this study, the effects of insertion and deletion (indel) mismatches distant from the target position on the gene correction were examined. Three target plasmid DNAs with and without indel mismatches â¼330 bases distant from the correction target position were prepared, and introduced into HeLa cells together with the TD. The indel mismatches improved the gene correction efficiency and specificity without sequence conversions at the indel mismatch site. These results suggested that the gene correction efficiency and specificity are increased when an appropriate second mismatch is introduced into the TD fragment.
