Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

Abstract

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

Fig. 1

Reaction scheme with variation of the antiprimer position. The gene is represented in blue, the vector backbone in gray, and the formed megaprimer in black. In the first stage of the PCR, both the mutagenic primer (positions randomized represented by a red square) and the antiprimer (or another mutagenic primer, shown to the right) anneal to the template and the amplified sequence is used as a megaprimer in the second stage. Finally, the template plasmids are digested using DpnI, and the resulting library is transformed in bacteria. The scheme to the left of the figure illustrates the three possible options in the choice of the megaprimer size for a single site randomization experiment. The scheme to the right represents an experiment with two sites simultaneously randomized

Fig. 2

Agarose gel analysis of the PCR amplification of pETM11-P450-BM3 (8474 bp) after DpnI digestion using the different protocols. Lanes 1 to 6 are F87 randomization experiments. Lanes 7 to 10 are M185/L188 simultaneous randomization experiments. 1 QuikChange™ protocol; 2 Kirsch and Joly protocol; 3 and 7 Zheng et al. protocol; 4 and 8 our protocol with small megaprimer; 5 and 9 our protocol with medium megaprimer; 6 and 10 our protocol with large megaprimer; M 1 kb DNA ladder standard (Fermentas)

Fig. 3

Gel electrophoresis of saturation mutagenesis reactions using pUCPCL6AN (6994 bp) after DpnI digestion containing the lipA gene from P. aeruginosa as template. Lane 1 small-sized megaprimer, clockwise; lane 2 medium-sized megaprimer, clockwise; lane 3 large-sized megaprimer, clockwise; lane M 1 kb DNA ladder standard (Fermentas); lane 4 small-sized megaprimer, anticlockwise; lane 5 medium-sized megaprimer, anticlockwise; lane 6 large-sized megaprimer, anticlockwise

Fig. 4

Our method applied to a plasmid template containing P. aeruginosa lipA gene (pUCPCL6AN). Different positions and orientations of the antiprimers were tried for the amplification of megaprimers both in the clockwise (a) and anticlockwise (b) directions. These different positions and directions encompass regions of the plasmid with differing GC content. Note that the mutagenic primer and only one of the antiprimers are used in each experiment

Fig. 5

Recommended working conditions. The amount of template and oligonucleotides are given for 50 μl of reaction mixture. The figure also depicts the three possible choices in the election of the antiprimer position