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.

Probing the equatorial groove of the hookworm protein and vaccine candidate antigen, Na-ASP-2

“…Using the cDNA of wild-type Na-ASP-2 in pPICZaA (Asojo et al, 2005) as template, site-directed mutagenesis based on the QuickChange/DpnI method (Zheng et al, 2004) was employed to generate the Na-ASP-2-H88A mutant. Complementary oligonucleotides carrying a single codon change (coding: 5'-GTA-TTC-AAG-GCC-TCG-CAA-CCT-AAC-CAA-AGG-AAA-GGA-TTG-GG-3', anti-coding: 5'-G-GTT-AGG-TTG-CGA-GGC-CTT-GAA-TAC-ACA-TTG-TTT-CGC-G-3') were used in a PCR reaction with PfuUltra II Fusion HS DNA Polymerase (Agilent, Mulgrave, Victoria, Australia).…”

Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis

“…To produce plasmid libraries that predominantly contained the desired site-saturation mutants, a minimum of aberrant plasmids, and as little template DNA as possible, the mutagenic primer pairs were redesigned according to the method of Zheng et al [15]. Briefly, the 5′-end of each primer was shortened and 3′-end lengthened relative to the mutation site, such that the forward and reverse primers were not perfectly overlapping and would bind preferentially to the template rather than to each other during annealing.…”

“…Attempts to construct plasmid libraries with mutagenic primers that were exact reverse complements except at the randomized positions, according to the standard Stratagene Quikchange protocol, resulted in an unacceptably high percentage of unproductive plasmids: template plasmids encoding cysteine, as well as insertion and frame shift mutants incorporating two copies of the primer sequence at the mutation site. Such aberrant clones are generally rare in Quikchange reactions not employing randomized codons and perhaps became abundant due to the necessity for the recipient bacteria to resolve mismatches between strands at the randomized positions.To produce plasmid libraries that predominantly contained the desired site-saturation mutants, a minimum of aberrant plasmids, and as little template DNA as possible, the mutagenic primer pairs were redesigned according to the method of Zheng et al [15]. Briefly, the 5′-end of each primer was shortened and 3′-end lengthened relative to the mutation site, such that the forward and reverse primers were not perfectly overlapping and would bind preferentially to the template rather than to each other during annealing.…”

“…However, using primers that were exact reverse complements, as the standard protocol dictates, made it nearly impossible to create a library containing a majority of non-aberrant mutants. Using primers that were not completely complementary [15] provided good coverage of all possible variants at the desired position, and decreased the number of aberrant plasmids obtained, but did not completely remove wild-type template plasmids from the reaction products, thereby complicating the use of the libraries for the positive genetic selection of functional mutants. However, modification of the Quikchange protocol to include a gel-purification step prior to bacterial transformation allowed the production of high quality plasmid libraries essentially free of unmutated template DNA and provided good coverage of all possible variants at the desired position, two conditions required for the success of this approach.…”

“…Identification of substitutions that are functional may not be straightforward in such cases, requiring a significant number of molecular biological and biochemical manipulations to construct and evaluate mutants that one thinks might be functional. Here we describe an unbiased and rapid approach that utilizes site-saturation mutagenesis based on the modified Stratagene Quikchange method of Zheng et al [15] to create libraries of mutants of the Crithidia fasciculata inosine/guanosine transporter gene, CfNT2, lacking one or more cysteine codons, and the subsequent genetic selection of highly functional transporter mutants in a heterologous expression system. The identification of functional mutants is greatly facilitated by the addition of a gel-purification step to the Quikchange protocol, which effectively eliminates background of wild-type clones in the selection.…”