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Description: JBS Mutagenesis Series 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 for superior function of the encoded proteins.
This example of natural evolution has guided researchers within the last two decades to develop strategies for in vitro permutation of proteins. Among the variety of strategies applied, three major powerful techniques have emerged.
→ JBS dNTP-Mutagenesis Kit #PP-101
The rate of mutagenesis achieved by error-prone PCR is in the range of 0.6-2.0%. → JBS Error-Prone Kit #PP-102
This method allows the recombination of sequences from different, related genes. The overall rate of mutagenesis is approx. 0.7%. → JBS DNA-Shuffling Kit #PP-103
Jena Bioscience now offers all components necessary for each of these techniques 'ready-to-go' in a separate kit, accompanied by a streamlined documentation that maximizes success.
Taq Polymerase (red cap)
5 units/μl, 40 μl
Mutagenesis Buffer (green cap)
10x concentration, 200 μl
dNTP Mix (white cap)
10 mM each dNTP (dATP, dCTP, dGTP,dTTP), 100 μl
dPTP (yellow cap)
10 mM, 40 μl
8-oxo-dGTP (blue cap)
10 mM, 40 μl
PCR-grade Water (white cap)
2x 1 ml
]. 8-Oxo-dGTP is incorporated opposite template adenine yielding two transition mutations: A→C:T→G.
A total rate of mutagenesis of approx. 2% (Fig. 2) has been reported, with A→C:T→G occurring at a ratio of approx. 1:1.5.
The dNTP analog dPTP has been reported to be approx. 10-fold more mutagenic than 8-oxo-dGTP (Fig.
2). The mutations induced by dPTP occur at a ratio of approximately 5:4:1:1 (A→G:T→C:G→A:C→T), with a total rate of mutagenesis of up to 19%.
Random mutagenesis induced by 8-Oxo-dGTP and/or dPTP is carried out in a two-step PCR process. First, the target DNA fragment is amplified in the presence of the four natural dNTPs plus the mutagenic analogs dPTP and/or 8-Oxo-dGTP.
The rate of mutagenesis can be easily controlled by the number of PCR-cycles (Fig. 2).
The product of the first PCR is then subjected to a second PCR in the absence of mutagenic analogs. This step eliminates the non-natural analogs from the target DNA before cloning and transformation.
Denaturation — 92°C — 1 min
Annealing 1) — 55-60°C — 1.5 min
Extension — 72°C — 5 min
Number of cycles: 5-30 (refer to Fig. 2) The rate of mutagenesis mainly depends on the number of cycles (Fig. 2) and can additionally be fine-tuned by the amount of mutagenic dNTP’s [Zaccolo et al.].
1) The annealing temperature depends on the melting temperature of the primers and should be adapted to the used primer set.
Fill up with PCR-grade Water to 50 μl. Use the same thermocycling conditions but 20-30 cycles.
