A new approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step
© Tyagi et al; licensee BioMed Central Ltd. 2004
Received: 20 September 2003
Accepted: 26 February 2004
Published: 26 February 2004
Site-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR.
The strategy relies on the use of a limiting concentration of one of the flanking primers (reverse or forward) along with the normal concentration of mutagenic primer, plus a prolonged final extension cycle in the first PCR amplification step. This first round of PCR generates a megaprimer that is used subsequently in the second round of PCR, along with the second flanking primer, but without the intermediate purification of the megaprimer. The strategy has been used successfully with four different plasmids to generate various mutants.
This strategy provides a very rapid, inexpensive and efficient approach to perform site-directed mutagenesis. The strategy provides an alternative to conventional megaprimer based site-directed mutagenesis, which is based on an intermediate gel purification step. The strategy gives a high frequency of mutagenesis.
Oligonucleotide-directed mutagenesis is one of the most popular methods to introduce site-specific alterations into a DNA sequence. In past few years, various protocols have been established for site-directed mutagenesis, which facilitate the analysis of gene expression and function. Currently, various PCR-based mutagenesis strategies are available but among them 'megaprimer'-based PCR has been used widely because of its simplicity and low cost .
Kammann et al.  originally developed the megaprimer-based mutagenesis strategy. Later, various changes have been suggested [3–7] to the original strategy. These megaprimer-based mutagenesis strategies are easy to use and they are very cost effective. The strategies require two rounds of PCR amplification using two flanking primers and one internal mutagenic primer. In the first round of PCR, one of the flanking primers and the internal mutagenic primer (having desired base substitutions) are used to generate a megaprimer. The megaprimer is purified by gel electrophoresis and extraction before being used along with the other flanking primer in the second round of PCR to generate the complete DNA sequence with the desired mutation.
Most of the reported megaprimer mutagenesis strategies rely on the intermediate purification of megaprimer after the first round of PCR amplification to remove the unused primers. This requires the use of cumbersome and time-consuming gel extraction steps. Three megaprimer based site-directed mutagenesis strategies are available at present that circumvent the gel purification step. The first one uses various restriction enzymes to cleave the first and second PCR templates and enzymatic removal of remaining primers from the first PCR reaction . Since this strategy is based on the use of DNA-modifying enzymes at various steps, it is complicated and expensive to use. In the second strategy the melting temperature (Tm) of the forward and reverse flanking primers play a critical role . A megaprimer is generated using a low Tm flanking primer and a low annealing temperature. The second PCR is carried out in the same tube and uses a high Tm flanking primer that prevents priming by the low Tm primer from the first PCR reaction, thereby obviating the need to gel-purify the megaprimer after the first PCR. However, the strategy is limited by the need to design primers in which one has a Tm that is substantially higher than the other; moreover, the average mutant frequency is approximately 80%. The third single tube strategy utilizes the three primers, including mutagenic primer, in three steps of PCR amplification to generate the mutant using different combinations of primers . The average mutagenesis frequency obtained with this strategy is about 50%. The complex, three-step PCR protocol, along with the lower average mutagenesis frequency, makes this strategy limited in use.
Our new strategy was applied to three other templates, using diagnostic restriction digestion to estimate the mutation efficiency without performing sequencing. In the case of the N346H mutation in the regulatory subunit of AHAS from A. thaliana , the second PCR product obtained using 0.05, 0.1 and 1.0 pmole of the first flanking primer, when digested with KpnI, showed different relative band intensity patterns when run on 1% agarose gel. The second PCR product obtained using 0.05 pmole of the first flanking primer showed only the bands corresponding to the anticipated mutant with no band of undigested (wild-type) product. In the 0.1 and 1.0 pmole experiments, a mixture of both wild-type and mutant was observed. The intensity of bands corresponding to mutant was very strong as compared to the wild-type for the 0.1 pmole experiment, while for the 1.0 pmole experiment an opposite banding pattern was observed. These results are consistent with our quantitative measurements on the KARI mutant. The strategy was also used to construct mutants of the regulatory subunit (D224A; Pham et al., unpublished) and catalytic subunit (M354V; ) of S. cerevisiae AHAS. The D224A mutagenic primer removes an EcoRV recognition site, while the M354V primer introduces a restriction site for ApaLI. Both mutations were tested using the same three different concentrations of first flanking primers. The results obtained from both of these mutants were in agreement with those obtained with the N346H mutant of A. thaliana AHAS.
The main reason for using a gel purification step after the first PCR in conventional megaprimer mutagenesis is to remove excess flanking primer. Without this step, the second PCR would contain both of the flanking primers and substantial amounts of wild-type product would be obtained. One of the novel features of our strategy (Fig. 1) is that the amount of the first flanking primer is limited, so that it is substantially depleted during the first PCR. This, in turn, leads to the second novel feature of our strategy, a prolonged extension step at the end of the first PCR. Because the amount of the first flanking primer is limited, a relatively small number of PCR cycles are sufficient to reach an end point. However, the small number of cycles constrains one of the essential features of PCR: full-length products accumulate exponentially while incomplete products accumulate arithmetically. Therefore, after just a few PCR cycles, full-length products will not overwhelm the partial products. We overcame this problem with a final and prolonged extension step, so that all of the partial products are converted to full-length products.
Mutations, primers and restriction enzyme recognition site added or removed.
Mutagenic primer sequence
E. coli KARI
A. thaliana AHAS Regulatory subunit
S. cerevisiae AHAS Regulatory subunit
S. cerevisiae AHAS Catalytic subunit
The results of sequencing experiments in the case of the KARI mutant, and diagnostic restriction digestion in the case of other three constructs, show the versatility of our strategy. Although there are currently three single PCR tube strategies available, ours is simpler and yields mutagenesis frequencies approaching 100%. We suggest that it could be applied to most constructs making it a simple but universal strategy for site-directed mutagenesis.
All PCR experiments were performed on a Perkin-Elmer 480 thermal cycler. Restriction enzymes, DNA ligase and DNA polymerase were obtained from New England Biolabs. Deoxyribonucleotide triphosphates (dNTPs) were purchased from Promega and the primers were obtained from Sigma-Aldrich Pty. Ltd. The mutagenic primers (Table 1) differed from the wild-type sequence by containing substitutions to change one amino acid residue. Mini preparation of plasmid DNA was performed using the Promega Plasmid Preparation Kit.
Our new strategy has been applied to four different proteins but for illustration the strategy to construct a mutant of E. coli KARI will be described in detail. Isolating clones followed by DNA sequencing was used to assess the success of the strategy. For the other three enzyme constructs, the designed mutagenic primers contained altered restriction enzyme recognition sites to permit facile screening for the mutant (Table 1). In one case; the target codon change was chosen so as to create a restriction enzyme recognition site, while in a second case an existing site was removed. A third mutagenic primer contained a second substitution that is silent with respect to the protein sequence but which introduced a restriction enzyme recognition site. The resulting mutants were screened using diagnostic restriction digestion.
The ilvC gene (1482 bp) encodes KARI and the gene product is a single polypeptide with a deduced molecular weight of 54 kDa . We have described previously the cloning of the gene into the pET30a(+) vector . The pET-C (6879 bp) plasmid was used as the template.
This new strategy consists of two rounds of PCR amplification. In the first round, the reaction was carried out in total volume of 50 μL and contained 2 U of Vent polymerase, 1X ThermoPol PCR buffer (10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.8 at 25°C), 2 mM MgSO4 and 0.1% Triton® X-100), 50–100 ng of pET-C DNA, 0.2 mM dNTP, 1.0 pmole of internal mutagenic forward primer (Table 1) and 0.05 pmole of the reverse flanking primer (T7 terminator, GGTTATGCTAGTTATTGCTCAGCGGTGGC). The PCR was carried out under the following conditions: denaturation, 95°C for 1 minute; annealing, 50°C for 30 seconds; and extension, 72°C for 2 minutes. This cycle was repeated 5 times and followed by an additional extension step at 72°C for 35 minutes. In the second round of PCR, the forward flanking primer (T7 promoter, CGCGAAATTAATACGACTCACTATAGGGG) was added into the same tube. After an initial denaturation of 96°C for 1 minute, 25 PCR cycles were used as follows: denaturation, 96°C for 1 minute; annealing, 55°C for 1 minute; and extension, 72°C for 2 minutes. These cycles were followed by an additional extension step at 72°C for 10 minutes. The second PCR product (1837 bp) having desired mutation was purified and digested with BamHI and HindIII to give a mutated ilvC gene fragment (1482 bp). The resulting fragment was purified and cloned into the expression vector pPROEX™HTb (4779 bp) (Gibco BRL) after digesting vector with BamHI and HindIII. The resulting mutant plasmid (6192 bp) was used to transform E. coli strain CU505 (Purdue University, Indiana, USA) competent cells and 10% of the transformed culture was cultured on Luria-Bertani agar plates supplemented with 10 μg/ml ampicillin. The plasmid isolated from selected colonies was sequenced using BigDye™ Terminator chemistry at the Australian Genome Research Facility (Brisbane, Queensland, Australia).
List of abbreviations
polymerase chain reaction
kilo base pairs
- Tm :
We thank Siew Siew Pang, Yu-Ting Lee and Sophia Pham for donating clones and oligonucleotides used in these studies. We also thank Linda Widjaja for assistance in preparing figures and tables. This work supported by the Australian Research Council, Project ID DP0208682.
- Ling MM, Robinson BH: Approaches to DNA mutagenesis: an overview. Anal Biochem. 1997, 254: 157-178. 10.1006/abio.1997.2428.View ArticleGoogle Scholar
- Kammann M, Laufs J, Schell J, Gronenborn B: Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR). Nucleic Acids Res. 1989, 17: 5404-View ArticleGoogle Scholar
- Sarkar G, Sommer SS: The "megaprimer" strategy of site-directed mutagenesis. BioTechniques. 1990, 8: 404-407.Google Scholar
- Landt O, Grunert HP, Hahn U: A general strategy for rapid site-directed mutagenesis using the polymerase chain reaction. Gene. 1990, 96: 125-128. 10.1016/0378-1119(90)90351-Q.View ArticleGoogle Scholar
- Brøns-Poulsen J, Petersen NE, Hørder M, Kristiansen K: An improved PCR-based strategy for site directed mutagenesis using megaprimers. Mol Cell Probes. 1998, 6: 345-348. 10.1006/mcpr.1998.0187.View ArticleGoogle Scholar
- Miyazaki K, Takenouchi M: Creating random mutagenesis libraries using megaprimer PCR of whole plasmid. BioTechniques. 2002, 33: 1033-1038.Google Scholar
- Lai R, Bekessy A, Chen CC, Walsh T, Barnard R: Megaprimer mutagenesis using very long primers. BioTechniques. 2003, 34: 52-56.Google Scholar
- Séraphin B, Kandels-Lewis S: An efficient PCR mutagenesis strategy without gel purification step that is amenable to automation. Nucleic Acids Res. 1996, 24: 3276-3277. 10.1093/nar/24.16.3276.View ArticleGoogle Scholar
- Ke S-H, Madison EL: Rapid and efficient site-directed mutagenesis by single-tube 'megaprimer' PCR strategy. Nucleic Acids Res. 1997, 25: 3371-3372. 10.1093/nar/25.16.3371.View ArticleGoogle Scholar
- Picard V, Ersdal-Badju E, Lu A, Bock SC: A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase. Nucleic Acids Res. 1994, 22: 2587-91.View ArticleGoogle Scholar
- Lee Y-T, Duggleby RG: Regulatory interactions in Arabidopsis thaliana acetohydroxyacid synthase. FEBS Lett. 2002, 512: 180-184. 10.1016/S0014-5793(02)02253-6.View ArticleGoogle Scholar
- Duggleby RG, Pang SS, Yu H, Guddat LW: Systematic characterization of mutations in yeast acetohydroxyacid synthase: Interpretation of herbicide-resistance data. Eur J Biochem. 2003,Google Scholar
- Barik S, Galinski MS: "Megaprimer" strategy of PCR: increased template concentration improves yield. BioTechniques. 1991, 10: 489-490.Google Scholar
- Wek RC, Hatfield GW: Nucleotide sequence and in vivo expression of the ilvY and ilvC genes in Escherichia coli K12. Transcription from divergent overlapping promoters. J Biol Chem. 1986, 261: 2441-2450.Google Scholar
- Hill CM, Duggleby RG: Purified recombinant Escherichia coli ketol-acid reductoisomerase is unsuitable for use in a coupled assay of acetohydroxyacid synthase activity due to an unexpected side reaction. Protein Expression Purif. 1991, 15: 57-61. 10.1006/prep.1998.0988.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.