title: Oligonucleotide site-directed mutagenesis in Xenopus egg extracts.
abstract: Addition of M13mp18 single-stranded DNA annealed with an oligonucleotide to a Xenopus egg extract results in a rapid and efficient incorporation of the oligonucleotide in a complete double-stranded supercoiled molecule. Both the efficiency of DNA synthesis and the recovery of complete double-stranded molecules are increased relative to the reaction carried out by the classical technique using the E. coli Klenow DNA polymerase, DNA ligase, dNTPs, ATP and ions. Site specific mutagenesis was assayed by reverting a point mutation in the lacz region of M13mp18. The color assay described by Messing and sequencing of the DNA extracted from isolated plaques was used to check for the reversion. A 2 hr incubation of the heteroduplex carrying the mutagenic oligonucleotide in the Klenow-ligase-dNTP mixture allows a recovery of 6% mutant phage after transformation of competent cells with the reaction products. Using the Xenopus egg extract, 83% mutant phage were recovered after the same incubation time, in reactions entirely performed in parallel. The Xenopus extract is stable and contains all components required for the assay, including all ionic and protein factors; thus the only addition is the annealed DNA. Such an eukaryotic system is therefore an attractive alternative to the reconstituted prokaryotic DNA polymerase-DNA ligase system for site specific mutagenesis.
mesh_terms: D000818:Animals; D001483:Base Sequence; D003090:Coliphages / Q000235:genetics; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D009865:Oocytes / Q000502:physiology*; D014020:Tissue Extracts / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D014020:Tissue Extracts