Multiple site mutagenesis with high targeting efficiency in one cloning step.

Site-directed mutagenesis is a widely used technique in molecular biology to generate single or multiple exchanges, insertions or deletions in DNA sequences. A number of strategies have been developed and used successfully (2,4). The QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) has proven valuable because it consistently yields high mutagenesis efficiencies and uses a rapid and simple protocol. The high-fidelity Pfu DNA polymerase (Stratagene) is used for PCR from a supercoiled plasmid template with two complementary mutagenesis oligonucleotide primers that minimize undesirable random mutations during PCR (8,9). Treatment of the DNA with restriction enzyme DpnI, which will cut only fully or hemimethylated 5′-Gm6ATC-3′ sequences in duplex DNA, leads to the selective digestion of the PCR template DNA (5,9). The in vitro synthesized unmethylated DNA, including the introduced base change(s), is resistant to DpnI digestion and used to transfect Escherichia coli competent cells. Because this preselection step eliminates the parental DNA, the ratio of mutation-harboring recombinant clones reaches levels above 95% for 2.9 kb templates and more than 83% for templates of 8 kb length (6). However, for the introduction of more than one point mutation at different sites, timeconsuming intermittent transformation and screening steps are required before the DNA template is available for a second round of PCR mutagenesis. Furthermore, it is often necessary to sequence the isolated recombinant clones to identify single base changes. This step substantially increases the time between consecutive rounds of mutagenesis. Since the introduction of the QuikChange site-directed mutagenesis method (6), variations of the basic protocol have been presented, including a procedure for simultaneous two-site mutagenesis using the megaprimer protocol (3). Here, we present a procedure based on the QuikChange mutagenesis system that allows the introduction of multiple mutations with high efficiency. By using in vitro dam-methylation between successive PCR amplifications, only a single transformation and DNA preparation step after the introduction of all mutations is required. A substantial amount of time is saved while still preserving high targeting efficiency. The β-galactosidase (lacZ) α-fragment gene from pUC19 (New England Biolabs, Beverly, MA, USA) carrying two or three point mutations (introduced by two or three sets of mutagenesis primers) was used as the template in our experiments to determine the mutagenesis efficiencies with our protocol. The construct harboring two mutations has a one-nucleotide insertion at a HindIII restriction site and a one-nucleotide substitution at a PvuII site. The template is referred to as pHmPm. The DNA construct with three point mutations carries an additional two-nucleotide deletion at an EcoRI site and is referred to as pEmHmPm. The corresponding oligonucleotide primer sets used for single-step mutagenesis were: Em1: 5′-GACGGCCAGTGAATTAGCTCGGTACCCGGG-3′ Em2: 5′-CCCGGGTACCGAGCTAATTCACTGGCCGTC-3′ Hm1: 5′-CTGCAGGCATGCAAGACTTGGCGTAATCATG-3′ Hm2: 5′-CATGATTACGCCAAGTCTTGCATGCCTGCAG-3′ Pm1: 5′-CACTTCGCTATTACGCTAGCTGGCGAAAGGG-3′ Pm2: 5′-CCCTTTCGCCAGCTAGCGTAATAGCGAAGTG-3′ All mutations generate premature stop codons, which gives rise to a truncated lacZ α-fragment that is not functional in complementation. To generate revertants from the mutant templates, two (pHmPm) or three (pEmHmPm) sets of primers were used for PCR with Pfu DNA polymerase. The respective reversion PCR primers were: Er1: 5′-GACGGCCAGTGAATTCGAGCTCGGTACCCGGG-3′ Er2: 5′-CCCGGGTACCGAGCTCGAATTCACTGGCCGTC-3′ Hr1: 5′-CTGCAGGCATGCAAGCTTGGCGTAATCATG-3′ Hr2: 5′-CATGATTACGCCAAGCTTGCATGCCTGCAG-3′ Pr1: 5′-CACTTCGCTATTACGCCAGCTGGCGAAAGGG-3′ Pr2: 5′-CCCTTTCGCCAGCTGGCGTAATAGCGAAGTG-3′ These primers were phosphorylated at their 5′ end with T4 polynucleotide kinase (New England Biolabs) as described (7). For all plasmid DNA preparations and transformations, we used the E. coli strain Epicurian Coli SURE (Stratagene). Figure 1 shows an overview of the protocol. The first PCR mutagenesis reaction with the pHmPm and pEmHmPm mutant templates was performed in a reaction volume of 50 μL that included 100 ng of template DNA, 150 ng (ca. 15 pmoles) of each primer, 200 μM dNTPs and 2.5 U of native Pfu DNA polymerase in 1× Pfu polymerase reaction buffer as provided by the supplier. For PCR mutagenesis, a total of 15 cycles were run with 30 s denaturation at 95°C, 1 min annealing at 55°C and 5 min 30 s extension at 68°C using the GeneAmp PCR Thermocycler System 9600 (PE Biosystems, Foster City, CA, USA). After restriction enzyme digestion with 10 U of DpnI for 1 h at 37°C, the DNA was electrophoresed in a 1.2% agarose gel to separate DpnI-resistant DNA. Using a QIAex II Gel Extraction Kit (Qiagen, Valencia, CA, USA), intact-size, undigested DNA was recovered from agarose gel slices. The isolated DNA was then incubated with 400 U of T4 DNA ligase, 10 U of T4 polynucleotide kinase and 10 U of E. coli Benchmarks