Nucleotídeos para Mutagênese Randômica

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.