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

Jae-Kyun Ko; Jianjie Ma

doi:10.1152/ajpcell.00517.2004

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

Jae-Kyun Ko·Jianjie Ma

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Abstract

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.

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Base Sequence Cell Physiological Phenomena DNA Primers Deoxyribonucleases, Type II Site-Specific Multigene Family Mutagenesis, Site-Directed Polymerase Chain Reaction Journal Article Research Support, N.I.H., Extramural Research Support, U.S. Gov't, P.H.S.

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Jae-Kyun Ko, Jianjie Ma. 2005-01-19. A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study.. https://doi.org/10.1152/ajpcell.00517.2004

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