Random mutagenesis by whole-plasmid PCR amplification

BioTechniques
A Parikh, F P Guengerich

Abstract

Random mutagenesis has become a powerful means of studying the effects of a large number of permutations of a particular DNA sequence. As a prime example, libraries of randomized cDNA clones, when translated into their corresponding proteins, can be useful in investigating the functional contributions of a mutagenized region to the overall properties of a protein. Existing molecular cloning techniques for random mutagenesis are tedious and frequently plagued with high levels of background from wild-type (nonmutagenized) template. We report a PCR-based method involving amplification of an entire plasmid containing a gene sequence of interest with partially complementary degenerate oligonucleotides for randomization of up to 12 consecutive nucleotide residues. Sequential treatment of the PCR product with Dpn/and a second specific restriction endonuclease and T4 DNA ligase followed by high-efficiency electroporation permits the generation of libraries with very low background. This technique should prove useful for studies on enzyme structure-function relationships as well as for other diverse applications.

Citations

Aug 29, 2002·Molecular Endocrinology·Rafael Vazquez-MartinezFredric R Boockfor
Oct 7, 2005·The Journal of Biological Chemistry·Donghak KimF Peter Guengerich
Mar 3, 2018·Cold Spring Harbor Protocols·Matteo ForloniNarendra Wajapeyee
Oct 11, 2005·The Journal of Biological Chemistry·Zhong-Liu WuF Peter Guengerich
Jan 4, 2001·Drug Metabolism Reviews·F P GuengerichE M Gillam
Apr 8, 2004·The Journal of Biological Chemistry·Melissa L Geddie, Ichiro Matsumura
Oct 25, 2001·Archives of Biochemistry and Biophysics·K NakamuraF P Guengerich
Apr 29, 2005·Biomolecular Engineering·Ichiro Matsumura, Lori A Rowe
Mar 24, 2007·Biochemical and Biophysical Research Communications·Takeshi SekiguchiJunko Fukumura
Sep 28, 2016·ACS Synthetic Biology·Pieter CoussementMarjan De Mey

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