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Journal of Biochemistry Advance Access published online on June 27, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn085
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© 2008 The Japanese Biochemical Society

Effects of target sequence and sense versus antisense 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, Japan; 2CREST, Japan Science and Technology, Japan; and 3Faculty of Medical Sciences, Kyushu University, Maidashi-3-1-1, Higashi-ku, Fukuoka 812-8582, Japan

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

Received May 9, 2008; Accepted June 16, 2008


  Abstract

Summary

The correction of an inactivated hygromycin-resistance and enhanced green fluorescence 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 antisense ss DNA fragments were prepared, and the gene correction efficiencies were determined by co-introduction of the target plasmid containing the gene with the ss DNA fragments. The gene correction efficiency varied (0.8-9.3%), depending on target positions and sense/antisense strands. Sense ss DNA fragments corrected the target gene with equal or higher efficiencies as compared to their antisense counterparts. The target positions corrected with high efficiency by the sense fragments also tended to be corrected efficiently by the antisense fragments. These results suggest that the sense ss DNA 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, single-stranded DNA fragment, nucleic acid therapeutics, genetic engineering, rpsL gene


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