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Cloning and Mutagenesis - Nucleotides for Random Mutagenesis

Random Mutagenesis is a common approach for directed evolution of proteins and for analysis of protein-structure/function relationships. In contrast to site-directed approaches, random mutagenesis is a promising tool for identification of beneficial mutations without prior structural and functional information about the protein of interest. Randomly introduced DNA mutations such as nucleotide exchange (substitution), insertion and deletion of one or multiple nucleotides result in amino acid sequence changes thus, producing proteins with altered characteristics such as enhanced or novel activities.

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A powerful approach for random mutagenesis is PCR-based incorporation of mutagenic nucleotide analogs into a DNA fragment[1-7]. The mutagenic potential of these nucleotide analogs relies on their alternate base pairing properties that lead to the introduction of several mutations during multiple rounds of a PCR reaction (Tab. 1). Elimination of the modified analogs is achieved by a second PCR in the presence of the four natural dNTPs, leaving highly mutated DNA ready for further investigation

Table 1: Nucleotide selection guide for random mutagenesis approaches.

Taq: Thermus aquaticus; transition: exchange of purine for purine (A ↔ G) or pyrimidine for pyrimidine (C ↔ T); transversion: exchange of purine for pyrimidine or vice versa (C/T ↔ A/G).

NucleotideMethod of incorporationPrimary type of mutationPrimarily induced mutation(s)Reference
8-Oxo-dGTPPCR (Taq Pol)TransversionA:T → C:G and T:A → G:C[1]
Combination of Error Prone-PCR (Mn2+) and PCR (Taq Pol) with 8-Oxo-dGTPTransversion
A:T → T:A and A:T → C:G
A:T → G:C and G:C → A:T
dPTPPCR (Taq Pol)TransitionA:T → G:C and G:C → A:T[1]
8-Oxo-dGTP & dPTPPCR (Taq Pol)Transversion
Mixture of mutations induced by single nucleotides[1,3]
5Br-dUTPPCR (Taq Pol)Transversion
A:T → G:C
T:A → C:G
2OH-dATPPCR (Taq Pol)Transversion
A:T → C:G and G:C → T:A
A:T → G:C
dITPPCR (Taq Pol)TransitionA:T → G:C and G:C → A:T[6,7,8]

Selected References

[1] Zaccolo et al. (1996) An Approach to Random Mutagenesis of DNA Using Mixtures of Triphosphate Derivatives of Nucleoside Analogues. Journal of Molecular Biology 255:589.
[2] Kamiya et al. (2007) Induction of various mutations during PCRs with Manganese and 8-Hydroxy-dGTP. Biol. Pharm. Bull. 30 (4):842.
[3] Zaccolo et al. (1999) The effect of high-frequency random mutagenesis on in vitro protein evolution: a study on TEM-1 beta-lactamase. Journal of Molecular Biology 285 (2):775.
[4] Ma et al. (2008) The mutagenic properties of BrdUTP in a random mutagenesis process. Mol. Biol. Rep. 35:663.
[5] Kamiya et al. (2004) Induction of transition and transversion mutations during random mutagenesis PCR by the addition of 2-Hydroxy-dATP. Biol. Pharm. Bull. 27 (5):621.
[6] Kuipers (2012) In vitro mutagenesis by using mixtures of dNTP and dITP in PCR. In: Methods of Molecular Biology 57. Humana Press.
[7] Wang et al. (2012) A simple and reproducible method for direct evolution: Combination of random mutation with dITP and DNA fragmentation with Endonuclease V. In: Molecular Biotechnology. Humana Press.
[8] Spee et al. (1993) Efficient random mutagenesis method with adjustable mutation frequency using PCR and dITP. Nucleic Acids Res. 21 (3):777.