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

✍ Scribed by Steve N. Slilaty; Margaret Fung; Shi-Hsiang Shen; Susan Lebel

• Publisher: Elsevier Science
• Year: 1990
• Tongue: English
• Weight: 767 KB
• Volume: 185
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(90)90279-i

✦ Synopsis

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.