Improved oligonudeotide site-directed rautagenesis using M13 vectors

Abstract

An improved method is described for the construction of mutations in M13 vectors using synthetic oligonucleotides. The DNA is first cloned into a novel M13 vector (based upon M13mp18 or M13mp19), which carries a genetic marker that can be selected against, such as an EcoK or EcoB site, or an amber mutation in an essential phage gene. In this "coupled priming" technique, one primer is used to construct the silent mutation of interest, and a second primer is used to eliminate the selectable marker on the minus strand. After primer extension and ligation, the heteroduplex DNA is transfected into a strain of E. coli which is repair deficient and selects against the plus strand marker. Over 50 mutants have been constructed with this approach, and the yields can be excellent (up to 70%). For the stepwise construction of mutations using separate rounds of mutagenesis, the EcoK and EcoB markers offer a particular advantage over the amber marker. They permit selection in each round, as it is possible to cycle between the two markers. However for construction of multiple mutations over a short region, long synthetic oligonucleotides with multiple mismatches to the template can offer an alternative strategy.

A survey of a functional amino acid of class Cβ‐lactamase corresponding to Glu166 of class A β‐lactamases

“…I. E. coli strains and plasmids E. coli TGl [17], a derivative of K12, was employed for DNA technology. E. coli AS226-51 [12], an ampD mutant of C600, which also has a deletion mutation in ampC, was used for measuring the antibiotic susceptibility of cells bearing the B-lactamase gene, and as host cells for enzyme preparation in order to avoid contamination by the ampC p-lactamase of E. coli.…”

Site-directed mutagenesis of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans

“…E. coli JMlOl is described in [14]. E. coli TG1 is an EcoK-derivative of JMlOl constructed by Toby Gibson at the MRC laboratory of Molecular Biology in Cambridge (UK), strain TG2 is a recA-version of TG1 (see [15]). …”

Site-Directed Mutagenesis

“…A disadvantage of these amber selection procedures (Carter et al, 1985b;Carter, 1986 Progeny phage derived from the plus (template) strand of M1 3 have been eliminated directly by preparing the template in a dut ung host strain of E. coli which results in few deoxyuridine residues being incorporated into the template in place of thymidine (Kunkel, 1985). After extension completely round the template from a mutagenic primer and ligation, the plus strand may be destroyed in vitro by treatment with uracil glycosylase and alkali.…”

“…Carter et al, 1985a,b;Carter, 1986 In constructing many mutations over a limited region, the synthesis of long oligonucleotides with multiple mismatches to the template provides an alternative strategy to the sequential construction of mutations. A 44mer with 13 mismatches to the template was used to direct seven simultaneous amino acid changes in the tyrosyl-tRNA synthetase of Bacillus stearothermophilus (Carter et al, 1985b). Deletions may be made using an oligonucleotide whose two ends are complementary to different regions of the template, so that in the annealing reaction the intervening region of template is looped out.…”