Process for site specific mutagenesis without phenotypic selection

US4873192A · kind A · utility

Assignee

• The United States of America, as represented by the Department of Health and Human Services · US

Inventor

• Thomas Kunkel · Ludwigshafen, DE

Key dates

• Filing date: May 18, 1988
• Grant date: Oct 10, 1989
• Expiry date: May 18, 2008

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/68
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

The present invention discloses several DNA mutagenesis processes using a DNA template containing several uracil residues in place of thymine, which can be applied without selection techniques to produce altered DNA sequences with approximately 10-fold greater efficiency than current methods of site-specific mutagenesis. This template has relatively normal coding potential in the in vitro reactions typical of standard site-directed mutagenesis protocols but is not biologically active upon transfection into a wild type (i.e., ung.sup.+) E. coli host cell. Expression of a desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus favored. The procedure has been applied to mutations introduced via both obligonucleotides and error-prone polymerization. The inclusion of two additional simple treatment steps before transfection results in a site-specific mutation frequency approaching 100%.