An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol
© Liu and Naismith. 2008
Received: 21 September 2008
Accepted: 04 December 2008
Published: 04 December 2008
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© Liu and Naismith. 2008
Received: 21 September 2008
Accepted: 04 December 2008
Published: 04 December 2008
Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable.
We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange™ site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis.
Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange™. Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange™) but increased the overall success rates.
Our new primer design scheme minimized the primer-primer dimerisation and enabled the primers to use the PCR products as the template. The schematic presentation of our new primer design is shown in Figure 2. Each primer pair contains non-overlapping sequences at their 3' end and primer-primer complementary (overlapping) sequences at the 5' end. The non-overlapping sequences are larger (significantly larger than suggested in ) than the complementary sequences to make the melting temperature of non-overlapping sequences (Tm no) 5 to 10°C higher than the melting temperature of primer-primer complementary sequences (Tm pp). The mutation sites can be placed either in the complementary region or non-overlapping region. Using the primers designed by our new scheme, the newly synthesized PCR products can be used as the templates in the subsequent PCR amplification cycles, which significantly increased the PCR efficiency and required less template DNA while it is unfeasible in the QuickChange™ or the modification reported previously .
Primers used for mutagenesis
Tm pp (°C)b
Tm no (°C)c
5'CATAATTTAATTCAAAAG CAC CAT C ACCATCATCACTGAGAATTCGAGCTCCGTCG3'
QuickChange™ site-directed mutagenesis has transformed the ability of labs to carry out site-directed mutagenesis. However its relatively low PCR amplification efficiency can lead to problems and the primer design needs care. Adding additional template alleviates this but can decrease the success rate for obtaining mutants as it leads to a increase of the amount of hemimethylated DNA molecules (newly synthesized strand combined with parental strand) which is resistant to DpnI digestion . Incomplete digestion results in recovery of non-mutated DNA. Modified primer design which destabilizes primer pairs has been proposed  to simplify the procedure. However, this and the original method can only use the parental DNA as template in the amplification cycles. This is because the primers either do not bridge or are too short to efficiently bridge the "nick" in the newly synthesized strands. By significantly increasing the non-overlapping region the primers should be long enough to bridge the "nick" and bind to the newly synthesized DNA and thus use it as template (Figure 1B, C) in the same way as normal PCR. We therefore designed primers which contain non-overlapping sequence and primer-primer complementary (overlapping) with a Tm no 5 to 10°C higher than the Tm pp so that the non-overlapping sequences can bridge the "nick" and bind to the newly synthesized DNA efficiently. These primer lengths are within the normal range suggested for QuikChange™ and therefore do not impose any additional costs to the laboratory. The PCR amplification using these primers showed high efficiency and as a result it required less than half the parental template and fewer amplification cycles. The decreased amount of parental DNA has the significant added benefit that decreases the amount of methylated and hemimethylated DNA which needs to be destroyed by DpnI, reducing the potential to recover the parental DNA.
The modified primer design as with that proposed earlier  eliminates the problems associated with primer pair self-annealing, and Tm values can be designed as these for conventional PCR . Moreover the restriction upon primer length is also lifted which enhances the utility of the technique. The removal of the primer length limitation allows adjacent multiple mutations to be made in a single step with a pair of mutagenesis primers without any limitation. We have successfully generated double mutations, double deletion and N-terminal deletion and C-terminal insertion mutants simultaneously in a single experiment, demonstrating that this modified method is efficient for multiple site-directed mutagenesis. PCR amplification for multiple-site mutagenesis produced partial DNA fragments with the forward primer in a primer pair (primer 1, Figure 1C) and the reverse primer of the downstream primer pair (primer 4, Figure 1C). These partial elongated DNA fragments annealed each other with their overlap sequences and extended to the full-length plasmid DNA in the subsequent PCR cycles  or functioned as the megaprimers in the subsequent cycles as described . There is no distance restriction of the mutation sites. However a long single pair primer should be used for the adjacent multiple mutations. In our experiments the double mutations (3026L/M-51L/M and 3327L/M-56L/M) spanned around 80 base pairs produced more full-length plasmid suggesting that the short partial PCR products could act as megaprimers in the subsequent amplification cycles more efficiently in comparison with the longer PCR fragments (VraRDNH/ICH and VraRDN3/DC5). An extra a few cycles using Tm pp-5 as the annealing temperature can increase the synthesis of the full-length plasmid and placing the mutation sites within the primer-primer overlap sequence can increase the mutation efficiency. Although Tm pp and Tm no of the primer pairs can be variable, a Tm no volume of the primer pairs 5°C to 10°C higher than Tm pp is required for an efficient PCR amplification. Under our PCR conditions, no plasmid concatemers were detected.
Our results demonstrated that the modified protocol is a high efficient method for single site mutagenesis and can be extended to multiple site-directed insertion deletion mutagenesis protocol without any extra steps such as ligation or phosphorylation.
As a result of this research, we have modified the site-directed mutagenesis protocol which increased the efficiency for single- and multiple-site mutations and also enabled facile large single insertions and deletions/truncations in a single experiment, an option incompatible with the standard QuikChange™ protocol or with the protocols reported previously [8, 12, 13, 17, 19, 25–27]. This single-step protocol utilized a new primer designing scheme and required significantly less parental DNA which facilitated its digestion after the PCR and enhanced the overall efficiency and reliability.
Six single-site mutations and three deletion mutants were generated using this modified protocol. The PCR amplifications with the primers designed by the new scheme revealed high amplification efficiency and required less parental DNA and PCR cycles. Sequence analysis the plasmid DNA revealed that in each mutagenesis reaction all four transformants contained the desired mutations or deletions. Four double-site mutations and two double-site deletions or deletion/insertion were generated using this method. A large number of recombinants demonstrated that our mutagenesis protocol was effective for engineering double mutations, deletions and insertions. Despite the fact that partial elongation products were produced and the syntheses of the full-length plasmid DNA variable, transformation of the resulting products into E. coli cells produced viable transformants. Three of four sequenced transformants contained the desired mutations.
Our new protocol has been used successfully to generate single and multiple-site mutations, deletions, insertion and combined insertion/deletions. The resulted mutants have been successfully used to express the proteins for structure determination (data not shown). The results demonstrated that this new protocol imposed no additional reagent costs but increased the overall success rates.
Plasmid pDESTSIRV30, pDESTSIRV33 expressing the SIRV proteins (CAG38830 and CAG38833), pDESTAVRA expressing MRSA vraR protein (CAG40961) and pDESTFaBH2 expressing Pseudomonas aeruginosa FaBH2 protein (AAG06721) were constructed using a modified Gateway technology with an N-terminal TEV protease cleavable His tag . All the plasmids were propagated in DH5α E. coli cells (Stratagene, La Jolla) and plasmids were prepared using Qiagen miniprep kits (Qiagen, Germany). Pfu DNA polymerase, DpnI restriction enzyme are provided with QuikChange™ kit purchased from Stratagene, additional Pfu DNA polymerase was purchased from Promega when required. All the primers were synthesized by Eurogentec and simply purified by SePOP desalting. The melting temperature was calculated as Tm = 81.5 + 16.6(log([K+]/(1+0.7 [K+])) + 0.41(% [G+C]) – 500/(probe length in base) – 1.0(%mismatch) . The Tm pp and Tm no were calculated for each primer. All primers and their Tm no and Tm pp are detailed in Table 1. PCR cycling was carried out using a Px2 thermal cycler (Thermo Electro Cooperation).
For single-site mutation, deletion or insertion, the PCR reaction of 50 μl contained 2–10 ng of template, 1 μM primer pair, 200 μM dNTPs and 3 units of Pfu DNA polymerase. The PCR cycles were initiated at 95°C for 5 minutes to denature the template DNA, followed by 12 amplification cycles. Each amplification cycle consisted of 95°C for 1 minute, Tm no -5°C for 1 minute and 72°C for 10 minutes or 15 minutes according to the length of the template constructs (about 500 bp per minute for Pfu DNA polymerase). The PCR cycles were finished with an annealing step at Tm pp-5 for 1 minute and an extension step at 72°C for 30 minutes. The PCR products were treated with 5 units of DpnI at 37°C for 2 hours and then 10 μl of each PCR reactions was analyzed by agarose gel electrophoresis. The full-length plasmid DNA was quantified by band density analysis against the 1636-bp band (equal to 10% of the mass applied to the gel) of the DNA ladders. An aliquot of 2 μl above PCR products, the PCR products generated using QuickChange™ or generated as described in  was transformed respectively into E. coli DH5α competent cells by heat shock. The transformed cells were spread on a Luria-Bertani (LB) plate containing antibiotics and incubated at 37°C over night. The number of colonies was counted and used as an indirect indication of PCR amplification efficiency. Four colonies from each plate were grown and the plasmid DNA was isolated. To verify the mutations, 500 ng of plasmid DNA was mixed with 50 pmole of T7 sequencing primer in a volume of 15 μl. DNA sequencing was carried out using the Sequencing Service, University of Dundee. For multiple site-directed mutations, deletions and insertions, the PCR was carried out in 50 μl of reaction containing 10 ng of template, 1 μM of each of the two primer pairs, 200 μM dNTPs and 3 units of Pfu DNA polymerase. The PCR cycles, DNA quantification, transformation and mutation verification were essentially the same as described above.
This work was carried out at Scottish Structural Proteomics Facility, which is funded by BBSRC, Scottish Funding Council (SFC) and the University of St. Andrews.
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