An efficient one-step site-directed and site-saturation mutagenesis protocol

Abstract

We have developed a new primer design method based on the QuickChange site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency. This design method minimizes primer dimerization and ensures the priority of primer-template annealing over primer self-pairing during the PCR. Several different multiple mutations (up to 7 bases) were successfully performed with this partial overlapping primer design in a variety of vectors ranging from 4 to 12 kb in length. In comparison, all attempts failed when using complete-overlapping primer pairs as recommended in the standard QuickChange protocol. Our protocol was further extended to site-saturation mutagenesis by introducing randomized codons. Our data indicated no specific sequence selection during library construction, with the randomized positions resulting in average occurrence of each base in each position. This method should be useful to facilitate the preparation of high-quality site saturation libraries.

Figure 1

Schematic diagrams of the partial overlapping primer design. Diamonds indicate the targeted mutation; additional silent mutations used for positive mutant screening by restriction endonuclease digestion are shown as triangles.

Figure 2

Agarose gel electrophoresis of mutagenesis reactions. Lane M: 1 kb DNA ladder standard (Fermentas). Lanes 1–4 use template pBAD-14D9: lane 1, D_H101A^*; lane 2, D_H101A; lane 3, A_L46T; and lane 4, F_H98A. Lanes 5–8 use template pBAD-10F11: lane 5, W_H104Y^*; lane 6, W_H104Y; lane 7, S_H100A; and lane 8, L_L101F. Lanes 9–11 use template pBAD-16E7: lane 9, D_H50A; lane 10, E_L39A; and lane 11, S_H99A; lane 12, F_H98A with template pQE-14D9ScFv; lane 13, F_H98A with template pET-14D9ScFv-NusA; and lane 14, CMV.

Figure 3

Agarose gel electrophoresis of site-directed saturation mutagenesis reactions. Lane M: 1 kb DNA ladder standard (Fermentas); lane 1, randomized PCR SSM1 with annealing temperature of 52°C; lane 2, randomized PCR SSM1 with annealing temperature of 57°C; lane 3, randomized PCR SSM2 with annealing temperature of 52°C; and randomized PCR SSM2 with annealing temperature of 57°C.

Figure 4

Sequencing results of site-directed saturation mutagenesis. Left, the PCR product (Figure 3, lane 1); Right, site-directed saturation library. Sequencing chromatography is represented using the software Chromas version 1.41. (http://trishul.sci.gu.edu.au/~conor/chromas.html/)