How does quick change mutagenesis work

Site-directed mutagenesis of Plasmids. A Generation of mutagenized plasmids. Upon PCR amplification, the template is removed by DpnI restriction digestion leaving behind only the mutagenized plasmid. Although it is commonly believed that the final products of the PCR reaction are pieces of double stranded, circular DNA with staggered nicks, recent data see Xia et al. These homologous ends recombine in vivo to give the final circular plasmid product pictured here.

B Screening of recovered plasmids for the mutation of interest. Similar results can be obtained with PCR screening using the primers specified by the arrowheads in A.

As a rule of thumb, 11 bp of complementary sequence on either side of the desired mutation usually mismatched bases is sufficient for your primers to successfully anneal to the plasmid of interest during the PCR reaction. The introduction or ablation of a restriction site through mutagenesis vastly facilitates the subsequent process of screening for succesfully mutated clones.

Forward and reverse primers are designed to be complementary, but each primer may extend beyond the complementary region as long as an overlap with a minimum 6 bp is maintained.

This overlap ensures that the PCR generates a nicked circle rather than a linear product see figure. A high purity plasmid prep significantly increases the success rate of site directed mutagenesis. In addition, you may want to try different concentrations of template e.

Be sure to adjust the extension time to match the size of your template. Since E. This was confirmed with the E. A The primers are annealed to the template DNA.

B The products of the first round of PCR are the same length as the plasmid. C In the following rounds of PCR, the newly synthesized strands are denatured and separated from the plasmid template.

E The overhangs were filled by the PCR enzyme. The gray cycles represent the parental plasmid DNA. Gray triangles indicate the location of the mutation. The new QuikChange TM mechanism has several implications. First, partially overlapping primers can be used to obtain the PCR product with homologous ends. The strategy has been successfully used to modify the QuikChange TM method by several groups 4 , 6 , 8— However, the use of partially overlapping primers is incompatible with the proposed mechanism.

Our findings support the use of partially overlapping primers because the products are the same whether completely or partially overlapping primers are used. The partially overlapping primers may be advantageous to minimize the formation of primer dimers.

Third, a small amount of template DNA is required for exponential amplification with partially overlapping primers, minimizing the template DNA surviving the DpnI digestion and yielding colonies containing the template plasmid. Fifth, E. With the new understanding, we tested Phusion with partially overlapping primers and a reduced amount of the template DNA.

The failure of Phusion to use complementary primers and primers with a long overlapping region in our experiments is likely due to the higher annealing temperature for Phusion PCR, which may favor the primer dimer formation Figure 3B , bottom instead of primer annealing to the template with mismatches.

A simple site-directed mutagenesis method using Phusion is developed. The method uses partially overlapping primers. The mutation site is preferentially at the middle of the overlapping region. Alternatively, electroporation can be used.

The new mechanism and method allow routine site-directed mutagenesis with commercially available reagents, and the method largely becomes a PCR issue. The advantages of using partially overlapping primers for the QuikChange TM method have previously been recognized 4 , 6 , 8—10 ; however, their use is incompatible with the proposed mechanism of the QuikChange TM method.

Here we provide evidence that the QuikChange TM mutagenesis proceeds through exponential amplification in vitro and homologous recombination in vivo with either completely or partially overlapping primers. Our findings provide the theoretical validation for using partially overlapping primers.

When using Phusion for the PCR, we recommend using partially overlapping primers. Phusion offers several advantages over PfuTurbo, being faster, more robust and with higher fidelity.

Our protocol does not need a ligation step. Google Scholar. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide.

Sign In or Create an Account. Sign In. Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract.

Yongzhen Xia , Yongzhen Xia. Oxford Academic. Wenqiao Chu. Qingsheng Qi. Revision received:. Select Format Select format. Permissions Icon Permissions. After you let it recover a bit you plate it on an agar plate in your classic Petri dish. Agar is related to but different from agarose and it makes a nice solid gel matrix to hold bacteria food. Usually we do this using antibiotic selection.

We use a plasmid that has an antibiotic resistance gene and then we spike the food with that antibiotic so that only bacteria with the plasmid can survive. So you have to put in a lot of the template. Once you have the changed DNA, the process is basically the same, no matter what the mutagenesis method.

Enzymes are usually proteins e. They bind to specific substrate s and provide the optimal conditions in their "active site" for a reaction to occur. So, after they help out with some reaction, they can do it again and again and again. I say "help out" because the enzyme doesn't really "do" anything - it can only help make something that was "possible," "likely. Instead, enzymes lower the activation energy required to get the reaction going.

This chops up the parent plasmid. But not the new one. So you can run a couple of controls. A summary showing the methods established for PCR-based site-directed mutagenesis. The products from overlap extension PCR were digested with restriction enzymes, and the resulting fragments were ligated with the plasmids digested with the same restriction enzymes.

The red stars in the left and right panels represent mutated bases. C A diagram for site-directed mutagenesis based on PCR with a pair of partially complementary primers, Dpn I digestion and transformation a or based on PCR with a pair of partially complementary DNA fragments, recombinational ligation, and transformation b. D A diagram for site-directed mutagenesis based on PCR with a pair of inverse primers, phosphorylation, ligation, and transformation.

The blue dots in the left panel represent phosphorylated groups. The red stars in A-D represent mutated bases. An effective method to avoid the formation of the primer dimers is that PCR for site-directed mutagenesis is performed using a pair of inverse primers 12 , This method has been confirmed to be effective for mutagenesis of small plasmids 12 , However, this method is time-consuming because PCR products have to be purified, phosphorylated, and ligated before the transformation.

Besides, the efficiency of DNA phosphorylation is usually so low that site-directed mutagenesis does not succeed at times even if PCR reactions are effective.

Apart from the methods described above, Edelheit and colleagues have reported a PCR-mediated method for site-directed mutagenesis, where two independent PCR reactions are performed using either forward or reverse single primers. The disadvantage of this method perhaps is yet the low efficiency for mutagenesis due to single primers used in PCR reactions.

Previous studies have shown that site-directed mutagenesis can be completed by combining PCR with DNA recombination techniques 15 , 16 , 17 , Kitagawa and Abdulle have reported a method for site-directed mutagenesis in vivo using three fragments from PCR and a homologous recombination system Recently, Trehan and colleagues have established a one-step method for site-directed mutagenesis using a bacterial system expressing a viral recombination protein and the products derived from PCR with a pair of partially complementary primers However, the bacterial system has to be prepared before mutagenesis.

Despite the availability of numerous methods for site-directed mutagenesis, mutagenesis of large plasmids remains difficult in vitro. In this study, we developed an effective method that can be applied to site-directed mutagenesis of large plasmids SMLP. We show that the SMLP method is highly efficient for the mutagenesis of the plasmids up to PCR-based methods for site-directed mutagenesis have been playing an important role in the generation of gene mutants in vitro.

We supposed that the reason for failure in the mutagenesis of Another reason is perhaps that the Pfu DNA polymerase Promega used in these assays was unable to amplify big-sized plasmids. To solve the problem of s ite-directed mutagenesis for large plasmids, we changed the strategy for PCR-mediated mutagenesis. PCR primers were designed based on the following guidelines: 1 Two pairs of partially complementary primers are designed and each pair of primers were separated and used in two independent PCR reactions.

A A plasmid map showing the locations of the PCR primers designed. The blue or red colours represent partially complementary overlap regions between two primers. The short black arrows represent the places digested by Exnase II; the long black arrows show how two DNA fragments are ligated. The resulting DNA fragments were mixed at the ratio of in moles where the amount of DNA for any of the fragments was not less than 30 ng.

Recombinational ligation was performed using an Exnase II kit as described in the section of the Methods Fig.

The colonies on the LB plates were selected, inoculated, and cultured overnight for plasmid preparation. The resulting plasmids were detected by agarose gel electrophoresis, and the plasmids with the correct size were verified by DNA sequencing.

From now on, we designated the method for site-directed mutagenesis of large plasmids as the SMLP method. Two independent PCR reactions were carried out as described in Fig. PCR products were purified and were then subjected to recombinational ligation. As expected, around one hundred and fifteen colonies were observed from one LB plate Fig. Next, we inoculated 5 mL LB medium by picking a single colony from the transformation plate; plasmids were prepared and detected by agarose gel electrophoresis.

These data indicate that the SMLP method can be applied to generating gene mutants for large plasmids. The SMLP method can be used for mutagenesis of the plasmid about The red line represents the mutated site in the plasmid.

The transformation plate was pictured using an EOS digital camera Canon. E Detection of the plasmids from the mutagenesis of MV by agarose gel electrophoresis. The DNA sequencing map for the MV mutant was presented in the upper panel in which a mutated base was framed by the red rectangle lines.

Both original and mutated DNA fragments were presented in the bottom panel, where the bases displayed in the sequencing map were outlined by the red rectangle lines.

The original and mutated bases were presented in green and red colours, respectively. To determine whether the result of mutagenesis for large plasmids is reproducible, we tried to generate different mutants for the pcDNA-FLNA plasmid using the same method as for the generation of MV mutant. These data indicate that the SMLP method can give rise to consistent results in site-directed mutagenesis of large plasmids. To substantiate this observation further, we tested the effect of the SMLP method on mutagenesis of large plasmids by increasing the distance between two pairs of primers used for PCR, where the small fragment was increased to 3.

Altogether, the SMLP method can produce consistent results during the mutagenesis of large plasmids although the distance between two pairs of primers has been changed. The SMLP method showed consistent results in the mutagenesis of large plasmids. PCR products were monitored by agarose gel electrophoresis. The plasmids from mutagenesis were prepared and analysed by agarose gel electrophoresis. The right panels are the original and mutated DNA sequences for each mutant, where the bases in the sequencing map were framed by the red rectangle lines.

The original and mutated bases were displayed in green and red colours, respectively. Alteration of the distance between two pairs of primers does not affect the mutagenesis efficacy of large plasmids. PCR products were detected by agarose gel electrophoresis. PCR products were detected as for B. D Analysis of the plasmids from site-directed mutagenesis by agarose gel electrophoresis. The plasmids derived from site-directed mutagenesis with the SMLP method were directly detected by agarose gel electrophoresis after plasmid preparation.

These plasmids displayed in a super-coiled form SL in the last lane or in a linear form the 3rd-5th lanes. The left panels are DNA sequencing maps for the AT and SL mutants, where the mutated bases are framed by the red rectangle lines. The right panels are the original and mutated DNA fragments for each mutant presented as for Fig.

So far we have generated several mutants from the The PCR products were then purified, and recombinational ligation and transformation were performed as done for the generation of the MV mutant. Plasmid detection and DNA sequencing confirmed the size and sequence of the DA mutant correct, indicating that the SMLP method can be applied to generating the mutants from the plasmids up to The SMLP method can be used for mutagenesis of the plasmids up to The primers were included in Table S2.

PCR products were detected by agarose gel electrophoresis as for Fig. C An image showing the result of transformation.