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Journal of Biochemistry Advance Access originally published online on June 27, 2008
Journal of Biochemistry 2008 144(4):431-436; doi:10.1093/jb/mvn085
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© 2008 The Japanese Biochemical Society.

Effects of Target Sequence and Sense versus Anti-sense Strands on Gene Correction with Single-stranded DNA Fragments

Hiroyuki Kamiya1,2,*, Masayuki Uchiyama1,2, Yoshimichi Nakatsu3, Teruhisa Tsuzuki3 and Hideyoshi Harashima1,2

1Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812; 2CREST, Japan Science and Technology; and 3Faculty of Medical Sciences, Kyushu University, Maidashi-3-1-1, Higashi-ku, Fukuoka 812-8582, Japan

*To whom correspondence should be addressed. Tel: +81-11-706-3733, Fax: +81-11-706-4879, E-mail: hirokam{at}pharm.hokudai.ac.jp

Received May 9, 2008; Accepted June 27, 2008


  Abstract

The correction of an inactivated hygromycin resistance and enhanced green fluorescent protein (Hyg–EGFP) fusion gene by a several hundred-base single-stranded (ss) DNA fragment has been reported. In this study, the effectiveness of this type of gene correction was examined for various positions in the rpsL gene. Sense and anti-sense ssDNA fragments were prepared, and the gene correction efficiencies were determined by co-introduction of the target plasmid containing the gene with the ssDNA fragments. The gene correction efficiency varied (0.8–9.3%), depending on target positions and sense/anti-sense strands. Sense ssDNA fragments corrected the target gene with equal or higher efficiencies as compared to their anti-sense counterparts. The target positions corrected with high efficiency by the sense fragments also tended to be corrected efficiently by the anti-sense fragments. These results suggest that the sense ssDNA fragments are useful for the correction of mutated genes. The variation in the correction efficiency may depend on the sequence of the target position in double-stranded DNA.

Key Words: gene correction, genetic engineering, nucleic acid therapeutics, rpsL gene, single-stranded DNA fragment

Abbreviations: ds, double-stranded; EGFP, enhanced green fluorescent protein; Hyg, hygromycin-resistance; ss, single-stranded


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