Rapid and Reliable Site-Directed Mutagenesis Using Kunkel’s Approach

  • Priya Handa
  • Swapna Thanedar
  • Umesh Varshney
Part of the Methods in Molecular Biology™ book series (MIMB, volume 182)

Abstract

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.

Keywords

Filtration Agar Urea Adenosine MgCl2 

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Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Priya Handa
    • 1
  • Swapna Thanedar
    • 2
  • Umesh Varshney
    • 2
  1. 1.Department of Microbiology and Cell BiologyIndian Institute of ScienceBangaloreIndia
  2. 2.Department of Cellular and Molecular Physiology, Penn State College of MedicineThe Pennsylvania State UniversityHershey

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