Kunkel method

Gene in plasmid with target site ( ) for mutation 

Denature the plasmid and anneal the oligonucleotide primers (J) containing the desired mutation (x) 

Parental DNA plasmid Mutagenic primer Mutated DNA plasmid 

Site-Specific Mutagenesis Using the QuikChange Kit from Stratagene 

An extension to this existing kit is the QuikChange Multi, which enables multiple mutations at different sites. It uses only one mutated oligonucleotide primer, in contrast to the kit described above, but follows the same procedure using the restriction enzyme Dpnl. The single-stranded plasmid is converted into a double strand in vivo after transformation into an E. coli host. 

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Fig. 1. Oligonucleotide-mediated, site-directed mutagenesis using the Kunkel method. See the text (sections on site-directed mutagenesis and mutagenesis with three primers) for further details, ss, Single-stranded.

H. Watzig, M. Degenhardt, and A. Kunkel, Strategies for capillary electrophoresis Method development and validation for pharmaceutical and biological applications, Electrophoresis, 19, 2695 (1998). 

Watzig, H., Degenhardt, M., and Kunkel, A. (1999). Strategies for capillary electrophoresis method development and validation for pharmaceutical and biological application. Electrophoresis 19, 2695-2742. 

Kunkel, A., Gunter, S., Dette, C., and Watzig, H. (1997). Quantitation of insulin by capillary electrophoresis and high-performance liquid chromatography — method comparison and validation. J. Chromatogr. A 781, 445—455. 

MF was first prepd in ihe 17th century by Swedish-German alchemist Baron Johann Kunkel von Lowenstern living from 1630 to 1703 (Hackh s Diet gives 1638 as date of birth). He obtd this dangerous expl by treating mercury with nitric acid and alcohol. The method was described in the book of Kunkel published in 1716 after his death. This book is listed and the prepn is briefly described by... 

A cassette-replacement approach was used to facilitate the introduction of amino acid mutations at various sites of the thrombin receptor. First, unique endonuclease restriction enzyme sites were generated at several positions within the thrombin receptor cDNA by mutating the nucleotide sequences. Second, the polymerase chain reaction (PCR) with primers encoding for the desired mutations was used to generate the cDNA cassette with the appropriate endonuclease restriction enzyme sites for replacement of the wild-type sequence. The locations for the introduction of the sites were chosen based on two requirements. They needed to be at or near regions of the cDNA sequence that codes for amino acids at junctions of transmembrane domains and extracellular loops. Also, introduction of the sites did not alter the amino acid sequence of the protein. The site-directed mutagenesis method of Kunkel et al.28 was used to introduce the mutations required for generating the... 

Kunkel, T. A., Bebenek, K., and McClary, J. (1991). Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol., 204, 125—139. 

At the close of the dialysis the plasma was centrifuged to remove small amounts of precipitate and the clear plasma resulting was subjected to starch-block preparative zone electrophoresis by the technique of Kunkel and Slater (15). At the close of the electrophoresis 1-cm. segments of the starch block were cut and transferred to sintered glass funnels, and the proteins were quantitatively eluted with five successive 2-ml. aliquots of 0.9% sodium chloride. The filtrates containing the protein fractions were then made up to a constant volume and mixed, and small aliquots were analyzed for total protein content by the method of Lowry et al. (18). 

Manahan, S.E., Kunkel, R. (1973). An atomic absorption analysis method for cyanide. Anal. Lett. 6 547-53. 

Evans BE, Rittle KE, Bock MG, DiPardo RM, Freidinger RM, Whitter WL, Lundell GF, Veber DF, Anderson PS, Chang RSL, Lotti VJ, Cerino DJ, Chen TB, KUng PJ, Kunkel KA, Springer JP, Hirshfield J. Methods for drag discovery development of potent, selective, orally effective cholecystokinin anatagonists. J. Med. Chem. 1988 31 2235-2246. 

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