The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA

Abstract

M13 RF IV DNA may be prepared in vitro to contain phosphorothioate-modified internucleotidic linkages in the (-)strand only. Certain restriction enzymes react with this modified DNA to hydrolyze the (+)strand exclusively when a phosphorothioate linkage occurs at the normal cleavage point in the (-)strand. The reaction of Pvu I with M13mp2 RF IV DNA containing dCMPS residues in the (-)strand is of this type, and is exploited to allow subsequent digestion with exonuclease III of a portion of the (+)strand opposite different mutagenic mismatched oligonucleotide primers. Two methods are described by which this approach has been used to produce mutations in M13mp2 phage DNA with high efficiency as a result of simple and rapid in vitro manipulations. Plaques containing mutant phage in a genetically-pure form are obtained at a frequency of 40-66%, allowing their characterisation directly by sequence analysis without prior screening and plaque purification. The wide applicability of this approach is discussed.

A mutant form of the rho protein can restore stress fibers and adhesion plaques in v-src transformed fibroblasts

“…This allows immunodetection of the rho protein (Munro and Pelham, 1987). Point mutations were introduced by subcloning the rho cDNAs into pTZ19 and then using site directed mutagenesis with the thioanalog method as described by Taylor et al (1985).…”

Transmembrane glutamic acid residues play essential roles in the metal‐tetracycline/H+ antiporter of Staphylococcus aureus

“…The method employed for mutagenesis was mainly that of Kunkel [24], however, for mutagenesis at codon 152, we employed the procedure of Taylor et al [25]. The mutagenic primers used were as follows: E30Q, 5'-ATTAAATCAAATGGTCCTAAATG-3'; E30D, 5'-TTAAATGA__CATGGTCCTAAATG*3'; E152Q, 5'-CTTTAGG-CCAAGGGTT-3'; E152D, 5'-TTAGGTGACGGCCTAGGTCCT-TC-3'; E397Q, 5'-TTTATCCCAAGGAACA-3'; E397D, 5'-TTAT-CAGACGGTACCGGTATAGC-3'.…”

Nucleotide induced conformation determines posttranslational isoprenylation of the ras related rab6 protein in insect cells

“…Secondary goat anti-mouse IgG coupled to peroxidase and [~-32P]GTP (3000 Ci/mmol) were from Amersham (Braunschweig, Germany) and [3H]mevalonolactone (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) Ci/mmol) from DupontNEN (Bad Homburg, Germany). Reticulocyte lysate was obtained from Serva (Heidelberg, Germany).…”

“…lB. Point mutations were introduced by subcloning the rab6 cDNAs into pTZ19 and by site-directed mutagenesis using the thioanalogue method as described by Taylor [21]. Wildtype and mutated cDNA were inserted into the pBlueBacHis A transfer vector (Invitrogen, San Diego) using the BamHI site.…”