© 2005 Oxford University Press
Conversion of nucleotide sequence with single-stranded DNA fragment prepared from phagemid DNA
1 Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Sapporo, 060-0812, Japan, 2 CREST, Japan Science and Technology, Japan, 3 Graduate School of Medicine, Tottori University, Nishi-machi 36-1, Yonago, Tottori 683-8504, Japan
The correction of a mutated gene is a highly attractive approach for gene therapy. This in vivo mutagenesis method will also be an effective tool in biotechnology. However, the current small fragment homologous replacement (SFHR) method with a heat-denatured double-stranded PCR fragment yielded the low correction efficiency. Single-stranded DNA fragments were prepared from single-stranded phagemid DNAs and tested in a gene correction assay with a Hyg-EGFP fusion gene inactivated by a substitution mutation, as a model target. A 606-nt sense, single-stranded DNA fragment dramatically (12–fold) improved the gene correction efficiency, although the antisense strand showed only minimal correction efficiency. On the other hand, correction of frameshift mutations with the sense single-stranded DNA fragment were 2-3–fold as efficient as that with the PCR fragment. These results suggest that the use of a sense, single-stranded DNA fragment is useful in the SFHR method for the correction of mutated genes.