Correction of frameshift mutations with single-stranded and double-stranded DNA fragments prepared from phagemid/plasmid DNAs.
We recently found that a heat-denatured, double-stranded DNA fragment, prepared from plasmid DNA (dsHES), and a sense single-stranded DNA fragment, prepared from single-stranded phagemid DNA (fSense), corrected an inactivated hygromycin-resistance and enhanced green fluorescence protein fusion (Hyg-EGFP) gene containing a base substitution (G:C to C:G) mutation 2-fold and more than 10-fold, respectively, more efficiently than the conventional PCR fragment (pcrHES), in the small fragment homologous replacement method. In this study, we tested the abilities of these new DNA fragments to correct Hyg-EGFP genes inactivated by one base insertion (+G) and deletion (-C) mutations. In contrast to its activity with the substitution mutation, the fSense fragment showed similar efficiencies to those of the dsHES fragment in the correction of frameshift mutations. For the correction of the insertion mutation, the efficiencies were in the order of dsHES (0.21%)>or=fSense (0.18%)>pcrHES (0.08%). In the case of the correction of the deletion mutation, the efficiencies were in the order of fSense (0.27%)>or=dsHES (0.19%)>pcrHES (0.12%). These results suggest that sense single- and double-stranded DNA fragments prepared from phagemid and plasmid DNAs, respectively, have the potential to correct frameshift mutations.
Tsuchiya
Hiroyuki
H
Graduate School of Pharmaceutical Sciences, Hokkaido University; Kita-12, Nishi-6, Sapporo, 060-0812, Japan.
Sawamura
Tomoko
T
Harashima
Hideyoshi
H
Kamiya
Hiroyuki
H
D000818:Animals; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D016368:Frameshift Mutation; D010957:Plasmids / Q000235:genetics*
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004279:DNA, Viral