Oligonucleotide-directed site-specific mutagenesis in Drosophila melanogaster.

AUTOR(ES)

Banga, S S

RESUMO

An efficient technique has been developed for performing in vivo site-directed mutagenesis in Drosophila melanogaster. This procedure involves directed repair of P-element-induced DNA lesions after injection of a modified DNA sequence into early embryos. An oligonucleotide of 50 base pairs, whose sequence spans the P-element insertion site, mediates base replacement in the endogenous gene. Restriction mapping, DNA sequencing, and polymerase chain reaction analysis demonstrate that base substitutions present in an injected oligonucleotide are incorporated into genomic sequences flanking a P insertion site in the white gene. This analysis suggests that progeny bearing directed mutations are recovered with a frequency of about 0.5 x 10(-3). Because Drosophila remains a premier organism for the analysis of eukaryotic gene regulation, this system should find strong application in that analysis as well as in the analysis of DNA recombination, conversion, repair, and mutagenesis.

Documentos Relacionados

• Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis.
• Oligonucleotide-directed site-specific integration of high complexity libraries into ssDNA templates
• High-efficiency oligonucleotide-directed plasmid mutagenesis.
• Predicting oligonucleotide-directed mutagenesis failures in protein engineering
• 5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis.