Improved gene correction efficiency with a tailed duplex DNA fragment.
A 606-base single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, corrects a hygromycin resistance and enhanced green fluorescent protein (Hyg-EGFP) fusion gene more efficiently than a PCR fragment, which is the conventional type of DNA fragment used in gene correction. Here, a tailed duplex, obtained by annealing an oligonucleotide to the ss DNA fragment, was used in the correction. The tailed duplex may be a good substrate for the RAD51 protein, an important enzyme in homologous recombination, which could be the gene correction pathway. The annealing of the oligonucleotides enhanced the correction efficiency of the Hyg-EGFP gene, especially when annealed in the 3'-region of the ss DNA fragment. Both the length and backbone structure of the oligonucleotides affected the gene correction efficiency. This type of gene correction device was also effective for another target gene, the rpsL gene. The results obtained in this study indicate that tailed duplex DNA fragments are effective nucleic acids for gene correction.
Tsuchiya
Hiroyuki
H
Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
Uchiyama
Masayuki
M
Hara
Kazuhiro
K
Nakatsu
Yoshimichi
Y
Tsuzuki
Teruhisa
T
Inoue
Hideo
H
Harashima
Hideyoshi
H
Kamiya
Hiroyuki
H
D000818:Animals; D016466:CHO Cells; D002934:Cinnamates / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004351:Drug Resistance / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000235:genetics; D052416:Targeted Gene Repair / Q000592:standards; D014158:Transcription, Genetic; D014162:Transfection
D002934:Cinnamates; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; C000717190:RpsI protein, E coli; C506766:RpsL protein, E coli; D049452:Green Fluorescent Proteins; D006921:Hygromycin B; C026273:hygromycin A; D051135:Rad51 Recombinase