Conversion of nucleotide sequence with single-stranded DNA fragment prepared from phagemid DNA.
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.
Tsuchiya
Hiroyuki
H
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Sapporo, 060-0812, Japan.
Sawamura
Tomoko
T
Uchiyama
Masayuki
M
Harashima
Hideyoshi
H
Kamiya
Hiroyuki
H
D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000737:chemistry*; D016368:Frameshift Mutation; D050939:Gene Fusion; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics
D004277:DNA, Single-Stranded; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins