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Am J Physiol Cell Physiol 288: C1273-C1278, 2005. First published January 19, 2005; doi:10.1152/ajpcell.00517.2004
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METHODS IN CELL PHYSIOLOGY

A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study

Jae-Kyun Ko and Jianjie Ma

Department of Physiology and Biophysics, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey

Submitted 25 October 2004 ; accepted in final form 17 January 2005

PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Herein we describe a rapid and highly efficient mutagenesis method using type IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type IIs restriction enzyme. After digestion of two fragments with a type IIs enzyme, exposed cohesive ends that are complementary to each other are then ligated together to generate a mutated gene. We applied this method to introduce multiple site-directed mutations in EGFP and Bcl-2 family genes and observed perfect mutagenesis efficiency at the desired sites. This efficient and cost-effective mutagenesis method can be applied to a wide variety of structural and functional studies in cell physiology.

Type IIs restriction enzyme; enhanced green fluorescent protein; Bcl-2


Address for reprint requests and other correspondence: J. Ma, Dept. of Physiology and Biophysics, Robert Wood Johnson Medical School, Univ. of Medicine and Dentistry of New Jersey, 675 Hoes Lane, 5th Floor, Research Tower, Piscataway, NJ 08854-5635 (E-mail: maj2{at}umdnj.edu)




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J.-K. Ko, K.-H. Choi, Z. Pan, P. Lin, N. Weisleder, C.-W. Kim, and J. Ma
The tail-anchoring domain of Bfl1 and HCCS1 targets mitochondrial membrane permeability to induce apoptosis
J. Cell Sci., August 15, 2007; 120(16): 2912 - 2923.
[Abstract] [Full Text] [PDF]


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