Rapid and Reliable Site-Directed Mutagenesis Using Kunkel’s Approach
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.
KeywordsFiltration Agar Urea Adenosine MgCl2
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