The Ligase-free Cloning Kit generates plasmids usable for transformation in only two simple steps:
1) Creation of a PCR product with ends homologous to the cloning vector.
2) Annealing and fusion of insert and vector in a circularization reaction.
No time consuming ligation step is necessary!
| Products & Ordering |
| Pfu-X Core Kit PCR-237 Kit for high accuracy PCR and Ligase-free cloning |
Within three billion years of evolution, nature has produced a plethora of proteins simply by repeated cycles of random mutagenesis followed by in vivo selection. This example of natural evolution has guided researchers to develop strategies for in vitro permutation of genes and proteins. Among a variety of strategies, three powerful techniques have emerged:
The method achieves mutagenicity of up to 20% and is based on incorporation of mutagenic dNTP analogs into an amplified DNA fragment. The analogs are eliminated by a second PCR in the presence of the four natural dNTPs, leaving highly mutated DNA ready for further investigation.
Mutagenesis is performed by a PCR reaction under modified conditions that induce an increased error-rate of the DNA-polymerase. Simply run the PCR protocol provided in the manual and achieve mutagenicity in the range of 0.6-2.0% in a single PCR reaction!
DNA shuffling generates libraries by random fragmentation of one gene or a pool of related genes, followed by reassembly of the fragments in a self-priming PCR reaction. The mutagenicity is similar to error-prone PCR but DNA shuffling allows recombination of sequences from different, related genes.
For each of these techniques we offer a ready-to-go kit, accompanied by detailed manuals and background information.
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.
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
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).
| Nucleotide | Method of incorporation | Primary type of mutation | Primarily induced mutation(s) | Reference |
| 8-Oxo-dGTP | PCR (Taq Pol) | Transversion | A:T → C:G and T:A → G:C | [1] |
| Combination of Error Prone-PCR (Mn2+) and PCR (Taq Pol) with 8-Oxo-dGTP | Transversion Transition | A:T → T:A and A:T → C:G A:T → G:C and G:C → A:T | [2] | |
| dPTP | PCR (Taq Pol) | Transition | A:T → G:C and G:C → A:T | [1] |
| 8-Oxo-dGTP & dPTP | PCR (Taq Pol) | Transversion Transition | Mixture of mutations induced by single nucleotides | [1,3] |
| 5Br-dUTP | PCR (Taq Pol) | Transversion Transition | A:T → G:C T:A → C:G | [4] |
| 2OH-dATP | PCR (Taq Pol) | Transversion Transition | A:T → C:G and G:C → T:A A:T → G:C | [5] |
| dITP | PCR (Taq Pol) | Transition | A:T → G:C and G:C → A:T | [6,7,8] |
[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.
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