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We describe a rapid and efficient polymerase chain reaction procedure for multi-site-directed mutagenesis for cases in which the sites to be mutated are in close proximity. The combination primer polymer chain reaction method is based on a multi-site directed mutagenesis protocol together with a splicing by overlapping extension polymerase chain reaction protocol. several different combinations of multiple mutations were successfully performed with this method and are reported in this study.
Polymerase chain reaction (PCR)–based oligonucleotide-directed site-specific mutagenesis is a technique extensively used to study protein structure-function relationships.1–3 Currently, few multi-site mutagenesis PCR-based strategies are available,4–6 but among them the QuickChange Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) has been used widely because of its simplicity. This kit is based on a thermal cycling step that denatures template DNA, anneals the multiple mutagenic primers, and extends and ligates these. The next step digests parental DNA, and a third step is transformation of the mutant single-stranded DNA into ultracompetent cells. Double-stranded plasmid DNA may then be prepared from the transformants and analyzed for desired mutations. One of the limits of these methods is the requirement of a specific proximity of the sites to be mutated. There is a physical limit (i.e., primer overlap/competition) to how close mutation sites can be. Others have tried to overcome this problem by designing primers containing multiple mutations.7 But as we have three different sites that we want to mutate in the seven possible combinations of single, double, and triple mutants, this would require separate primer designs for all desired combinations (i.e., seven different primers in two orientations).
We here describe a new strategy to make a whole range of different multimutagenic primers by using single-mutagenic primers as both template and primers. The new strategy requires two rounds of PCR amplification using two partially complementary overlapping mutagenic primers. In the first round of PCR, the primers are combined, resulting in a mix of newly synthesized multimutagenic primer “dimers” along with the single-mutagenic primers. This multiprimer mix from the first round is used without purification in the second round of PCR to generate the DNA sequence with the desired mutations. The product from the second round of PCR is transformed directly, and without purification, into cells, and plasmid DNA from the transformants is screened for mutations by sequencing. We tested our new strategy by making a variety of recombinant human mannan-binding lectin (rhMBL) mutants. The objective was to make cysteine-to-serine mutations at positions 25, 32, and 38 in all possible combinations (i.e., three single mutants, three double mutants and the triple mutant. Figure 11 clearly illustrates the close proximity of the sites to be mutated.
Plasmid pME721-forMBL, encoding the 71 N-terminal amino acids of human MBL (GeneBank Accession No. X15422) in the vector pGEM11fZ- (Promega) served as template. A QIAprep Miniprep Kit (Qiagen 12125) was used to prepare the double-stranded plasmid template. Oligonucleotide primers were synthesized at 40-nmol scale and purified by standard desalting by the manufacturer (DNA Technologies A/S, Aarhus, Denmark) without further purification. For all primers, mutated bases are marked in bold, and sense and antisense orientations are denoted as sb and ab, respectively. The name reflects the position of the cysteine to be mutated to a serine:
Setup 1 (Figure 22 and Table 11)) consists of two traditional QuickChange Multi Site-directed Mutagenesis PCR reactions set up with one (setup 1B) or two (setup 1A) primers and using plasmid DNA as template.
Setup 2 (Figure 33 and Table 11)) consists of two consecutive sets of PCR reactions. First, two reactions were set up, one containing C25Ssb (sense) and C32Sab (antisense; setup 2A) and the other containing C32Ssb and C38Sab (setup 2B), both without using DNA template. In the second set of PCR reactions, the unpurified products of the first PCR reactions were added as primers using plasmid DNA as template.
In setup 3 (Figure 44 and Table 11),), a PCR using a mixture of the two unpurified double-mutant products resulting from the previous step (first PCR, setup 2, marked with bold in Table 11 and red in Figures 33 and 44)) as template, and the two outmost primers, C25Ssb and C38Sab, was set up. The unpurified product of this reaction was used as primers in a second reaction with plasmid DNA as template.
PCR amplifications were performed on a BioRad i-cycler. The five reactions using plasmid DNA as template were all 25-μL reactions containing 100 ng plasmid DNA template and approximately 100 pmol of the appropriate primer (in the cases where the primer concentration is known). The reactions were performed using the QuickChange Multi Site-Directed Mutagenesis Kit (Stratagene 200514) using the buffers, enzymes, and dNTP mix provided. The amplification was performed for 30 cycles using the following conditions: 95°C for 1 min, 55°C for 1 min, and 65°C for 7 min. The primer annealing and amplification reactions described in setup 2; first PCR were 20-μL reactions containing approximately 100 pmol primer and 10 μL PfuUltraHotstart PCR 2X Master Mix. The reactions were performed using PfuUltraHotstart PCR Master Mix Kit (Stratagene 600630). The amplification was performed for 5 cycles under the conditions: 95°C for 30 sec and 72°C for 1 min. Afterwards, 10 cycles were performed under the conditions: 95°C for 30 sec, 66°C for 30 sec, and 72°C for 1 min. Then 25 cycles under the conditions: 95°C for 30 sec, 61°C for 30 sec, and 72°C for 1 min, followed by a final extension step for 10 min at 72°C. One microliter of each of the two reaction products were mixed with 8 μL H2O, and 1 μL of this mix was used as template in the first PCR reaction described in setup 3: A 20-μL reaction containing the 1-μL template, approximately 100 pmol of each primer and 10 μL PfuUltraHotstart PCR 2X Master Mix. The amplification was performed as described for the primer annealing reactions in setup 2. The five final PCR products were transformed directly and without purification, into the Ultra competent cells of the QuickChange Multi Site-directed Mutagenesis Kit and plated onto Lauria-Bertani + ampicillin plates. Isolating clones and screening by DNA sequencing (performed by LARK Technologies, Essex, UK) was used to assess the success of the strategy, and the mutation frequencies are given in Table 11.
Setup 1 yielded the three single mutants and a double mutant, C25S-C38S. The mutation frequencies were 37.5% for the C25S mutant, 12.5% for the C38S and the C25S-C38S mutants, and 25% for the C32S mutant.
Setup 2 involved two sets of two consecutive PCRs. The first set of PCRs was the production of combinational primers by annealing and amplifying adjacent sense (sb) primers and antisense (ab) primers to overcome primer competition due to close proximity of mutation sites. This was performed using the C25Ssb-C32Sab combination and the C32Ssb-C38Sab combination. The second set of PCRs used the unpurified mix of new primers on the plasmid DNA template. These two sets of reactions yielded both the single mutant C32S and the two remaining double mutants, C25S-C32S and C32S-C38S. The mutation frequencies were 40 and 16.7% for the C32S mutant in the two different reactions, 20% for the C25S-C32S mutant, and 33.3% for the C32S-C38S mutant.
Setup 3 also involved two consecutive PCRs and was an attempt to produce the triple mutant. The first PCR was again the production of combinatorial primers using the double-mutant primers obtained in setup 2, PCR1 as template for the flanking primers (C25Ssb and C38Sab) to obtain the triple-mutant primers. The second PCR again used this unpurified primer mix on the plasmid DNA template. This reaction yielded a mix of mutants containing the single, double, and most importantly the triple mutant. The mutation frequencies were 12.5% for the C38S and the C25S-C32S-C38S mutants and 25% for the C32S-C38S mutant.
All the results are summarized in Table 11.
The challenge was making C25S, C32S, and C38S mutations of rhMBL. The aim was to obtain the three single mutants, the three double mutants, and the triple mutant. The sites to be mutated were separated by 20 nucleotides and 16 nucleotides, respectively. Thus it was impossible to design three well-separated mutagenesis primers for each mutation to be achieved, which is required if the QuickChange Multi Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) or the method of Seyfang and Jin8 is to be used. The other multi-site mutagenesis PCR-based strategies are limited by the number of sites that can be mutated6 or the distance between the sites to be mutated,4 or are very laborious.5 Also, the Zheng et al. approach7 would pose some problems. First, we would have to design all the seven different primers, sense and antisense, corresponding to the seven different mutants we want to make. This would require 14 different primers. Second, the primers would have to be >55 bp long, which would lead to primer dimerization as opposed to generation of mutants.9
We therefore tested the idea of generating “combination primers” by overlapping PCR. Subsequently, we used these unpurified combination primers directly in mutagenesis PCRs as primers on plasmid DNA template. The PCR products were transformed directly, without purification, and the transformants were screened for mutations by sequencing of plasmid DNA. The strategy was successful. We managed to obtain all the possible combinations of mutants in a fairly simple setup and with convincing mutation frequencies.
The results of sequencing experiments show the versatility of our strategy. It is simple and yields all combinations of mutants at high frequencies. We suggest that it could be applied to most constructs, making it a simple but universal strategy for close proximity multisite-directed mutagenesis.
NatImmune A/S and The Danish Ministry of Science, Technology and Innovation are gratefully acknowledged for financial contributions.