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Site-directed mutagenesis by complementary-strand synthesis using a closing oligonucleotide and double-stranded DNA templates.

S N Slilaty·M Fung·S H Shen·S Lebel
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Abstract

An approach for generating structures capable of directing full-length complementary-strand synthesis for double-stranded plasmid DNA is described. The structures are formed following heat denaturation and cooling of linearized plasmid DNA molecules in the presence of what is referred to as a "closing" oligonucleotide. Consisting of a sequence complementary to the free ends of one of the two plasmid strands, the closing oligonucleotide functions as an agent for recircularization of a DNA strand and generation of a primer-circular template structure suitable for polymerase-dependent full-length complementary-strand synthesis and ligation into a covalently closed heteroduplex molecule. When combined with a mutagenic oligonucleotide and uracil-substituted DNA templates, this approach allows site-directed mutagenesis to be performed directly on double-stranded DNA with a mutant formation efficiency of about 50%, a level amenable to rapid screening by DNA sequencing.

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Base SequenceDNADNA, BacterialDNA-Directed DNA PolymeraseEscherichia coliMolecular Sequence DataMutationOligonucleotidesPlasmidsRestriction MappingTemplates, GeneticTransformation, Bacterialbeta-GalactosidaseJournal Article

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S N Slilaty, M Fung, S H Shen, S Lebel. 1990-02-15. Site-directed mutagenesis by complementary-strand synthesis using a closing oligonucleotide and double-stranded DNA templates.. https://doi.org/10.1016/0003-2697(90)90279-i

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