Rapid and Reliable Site-Directed Mutagenesis Using Kunkel’s Approach
| Abstract |
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Site-specific mutagenesis is a powerful tool in molecular biology research. A number of techniques are available today for
carrying out site-directed mutagenesis (SDM). Common among them is the oligonucleotide-directed mutagenesis (1). Three widely used procedures, which are based on this principle, are the polymerase chian reaction (PCR)-based approach
(2), and Kunkel’s (3) and Eckstein’s (4) methods. Kunkel’s method, which takes advantage of a strong biological selection, although inexpensive, has a drawback,
in that its efficiency of selecting against the wild-type parent strand from the heteroduplex is not efficient. In addition,
the enzymes used in this procedure are contaminated with uracil DNA glycosylase (UDG), which may also contribute to the overall
low efficiency of mutagenesis.
Affiliation(s): (2) Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
(3) Department of Cellular and Molecular Physiology, Penn State College of Medicine, The Pennsylvania State University, Hershey, PA
(3) Department of Cellular and Molecular Physiology, Penn State College of Medicine, The Pennsylvania State University, Hershey, PA
Book Title: In Vitro Mutagenesis Protocols
Series: Methods in Molecular Biology | Volume: 182 | Pub. Date: Nov-06-2001 | Page Range: 1-6 | DOI: 10.1385/1-59259-194-9:001
Subject: Genetics/Genomics
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