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This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted nobagen, distribution, and reproduction in any medium, provided the original work is properly cited. As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties i.
Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study.
We have measured the error rates for 6 DNA polymerases commonly used in Novagej applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products.
The strategy employed here allows interrogation of error rate across a very large DNA sequence kor, since 94 unique DNA targets were used as templates for PCR cloning.
Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, lod little bias observed for type of transition.
With the rapid pace of developments in systems biology-based research, for example, genomics, proteomics, and metabolomics, larger-scale biological discovery projects are becoming more common. An example of research that has been transformed by developments in systems biology is the cloning of expressed open reading frames ORFs from cDNA substrates.
The traditional path for ORF cloning has usually started with experimental novsgen driving the identification of one or several genes of interest to a particular pathway. Cloning novaben target s then typically resulted in further refinements of pathway details and often identification of new cloning targets.
With the creation and continual refinements of databases of genomic sequences, cloning now often takes place on a much larger scale. Microarray technology and DNA sequencing breakthroughs have led to a vast increase in the number of ORFs present in biological databases. Furthermore, biological observations no longer necessarily precede target identification, which now is often driven in large part by bioinformatics-based predictions and analyses.
Examples of large-scale cloning efforts include structural genomics projects to systematically determine protein structures [ 1 ], pathogen ORF cloning to understand disease and therapeutic mechanisms [ 2 ], and creation of the entire human ORFeome which will further developments in basic and applied biomedical sciences [ 3 ].
Minimizing PCR-generated errors is especially important for larger-scale cloning projects because, given a sufficiently large pool of target DNA sequence, even high fidelity enzymes will produce clones with mutations. There are a variety of methods to assay the fidelity of a DNA polymerase. Direct sequencing of clones was a practical approach at the time due to the low fidelity of the polymerase; that is, most clones that were sequenced would contain at least one mutation.
With the introduction of higher fidelity polymerases, new screening methods were developed to rapidly interrogate large numbers of PCR products for the presence of mutations. These assays were based on a forward mutation fidelity assay developed by Kunkel and colleagues, which used a gap-filling reaction with a DNA polymerase on a lacZ template sequence, followed by ligation and transformation into E.
Colorimetric screening based on a functional lacZ gene allowed rapid identification of mutations, which were subsequently sequenced to determine the nature of the DNA alteration [ 7 ]. This method, sometimes using a different reporter gene, has been used to screen a variety of high fidelity PCR enzymes and to optimize PCR reaction conditions to minimize mutations [ 48 ].
Finally, methods that rely on assaying PCR mutations based on differing chemical properties i. While reported fidelity values differ among research groups and assay methods, there is a general consensus that a relatively low-fidelity enzyme such as Taq has a fidelity value in the range and higher fidelity enzymes have values that are in the range usually reported as mutations per bp per template doubling.
A tradeoff involved in using screening methods like those described above is that generally only one DNA sequence is interrogated during the assay. Additionally, limitations built into the assays further restrict the possible mutations that can be detected. For example, the assay based on screening lacZ gene amplification products uses a single 1. Because polymerase errors are known to be strongly dependent on DNA sequence context reviewed in [ 12 ]ideally one would use a large set of DNA sequences when measuring enzyme fidelity.
This becomes especially relevant in the context of large-scale cloning projects, which involve hundreds or thousands of targets and thus contain an almost infinite DNA sequence space. To this end, we have designed and executed a study that measures enzyme fidelity by direct sequencing of cloned PCR products. Falling costs for DNA sequencing have made this method of fidelity determination practical, even for enzymes that make few mistakes.
Our goals are to compare fidelity values derived from direct clone sequencing to those derived from screening-based methods, as well as to evaluate these results in the context of choosing an enzyme for a high-throughput cloning project. To determine error rates and observe mutational spectra for a variety of DNA polymerases used in PCR cloning, we directly sequenced clones produced from 94 different plasmid templates.
These plasmids, each with a unique target DNA sequence, are a subset of a larger group of glycosyltransferase clones that we have prepared from Arabidopsis thaliana cDNA manuscript in preparation. A summary of the 6 DNA polymerases used in this study is presented in Table 1.
We included Taq polymerase in our study because of the extensive body of literature that exists on the fidelity properties of this enzyme.
Our cloning pipeline uses recombinational insertion of purified PCR products into a plasmid vector using the Gateway cloning system, a method widely used for high-throughput cloning studies reviewed in [ 17 ]. Since our input plasmid DNA templates were prepared using the Gateway system, the target genes of interest are all flanked by att recombination sequences.
This allowed the use of common primers for all PCR polymmerase, thus eliminating the need for target-specific optimizations. Figure 1 shows gel images for a representative set of PCR reactions for each enzyme. In all cases, a single major product band migrating at the expected size was observed. Amplification efficiency kor measured by quantitation of PCR product using a dsDNA-specific dye and calculating the fold-amplification based on a known quantity of input DNA template.
The fold-amplification is used to determine the number of template doublings that occurred during PCR. Kdo reported in Table 2amplification efficiency values were fairly uniform for all samples within a plate. We observe similar amplification efficiencies between different enzymes, with the exception that we routinely observed fewer template doublings in reactions with Pfu polymerase.
We have kept thermocycling protocols constant for all enzymes, and thus it is possible that some parameters were not optimal for amplification by Pfu.
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This precipitation step can be performed in well plate format, which is a requirement when the number of samples becomes large. Three colonies per plate were picked and grown polumerase in well plates, and cultures were screened for correct-size insert by colony PCR. For each target, one or more clones for each target containing a correct-size insert if obtained were cultured and used for DNA sequencing. As an early workhorse in PCR technology, Taq polymerase has been studied extensively for purposes of fidelity determination.
The type and number of individual mutations are listed in Table 3. This value is in excellent agreement with other published values for this enzyme, and the relatively high variance suggests that calculated error npvagen differing by up to 2-fold are probably not significant relative to the experimental noise. Polynerase, the spectrum of the base substitution mutations agrees well with previous observations on Taq polymerase reported in the literature [ 7 ].
There was only one insertion or deletion indel mutation observed in our data set, a single T deletion in a template sequence. Both elevated magnesium and dNTP levels were subsequently shown to elevate frameshift indel mutations preferentially relative to base substitution mutations [ 21 ].
An important control for these experiments is necessitated by the method used to generate template for DNA sequencing. For larger-scale cloning projects, DNA sequencing using cell culture is advantageous because of the saving in time and resources relative to purifying plasmid DNA. Every one of the fourteen mutations detected in the subset using cell culture as the source for sequencing template was also observed when sequencing from plasmid DNA template data not shown. Furthermore, based on our results with Taq polymerase, we conclude that our method for fidelity determination gives results in excellent agreement with other studies and is thus an accurate measure of polymerase accuracy.
Our results indicate that 3 of the enzymes included ;olymerase the study, Pfu polymerase, Phusion Hot Start, and Pwo polymerase, have error rates that are significantly lower than the others. This is consistent with previous findings demonstrating very high fidelity PCR amplification for these enzymes. The slight error frequency value differences are probably not significant, given that the small number of mutations is produced by these high pilymerase polymerases in addition to the experimental variability discussed above for the results with Taq.
Given the costs of cloning and sequencing and finite research budgets, mutation detection by DNA sequencing of clones generates a relatively small data set of mutations when the enzyme fidelity is high.
This is a drawback to our assay, and despite the fact that DNA sequencing costs continue to drop screening bacteria is still a far more economical method of interrogating a large number of clones. For all mutant clones produced by PfuPhusion Hot Start, and Pwo okd, samples were resequenced to rule out sample processing or DNA sequencing as a source of error.
In all cases, the original mutation was present, confirming the PCR reaction as the most likely source of the mutation. From the standpoint of use in a large-scale cloning project, any one of these enzymes would be acceptable, judged on the criteria of minimizing error rate. Other factors need to be considered of course, such as amplification efficiency, mutation spectra, performance with high GC content templates, and cost, to name a few.
Both indel mutations occurred in repeat regions, with one being an A insertion into an template sequence and the other being a Novqgen deletion within a template sequence. This result was unexpected in light of the high processivity of Phusion polymerase relative to other commonly used PCR enzymes vendor website.
Because multiple studies have found that increased polymerase processivity reduces the frequency of slippage mutations that result in indel nvagen [ 2223 ], we expected Phusion to produce the fewest of this class of errors.
It should be noted, however, that this conclusion is based on a small sample size and a larger number of mutations should be analyzed for confirmation. For the study of Phusion fidelity, the PCR used a different buffer than the one employed here, which according to the vendor does result in a fold lower error rate.
In addition, that study uses the BEAMING method, an extremely sensitive flow cytometric protocol that screens large numbers of beads that contain PCR products for the presence of nucleotide variations.
Thus, while the assay is extremely sensitive for detection of defined mutations, results kld with the BEAMING method for mutation frequency at a single position may not necessarily reflect the fidelity properties of an enzyme for much larger sequence spaces.
For the study on Pfu error rate, several fundamental methodological differences are present: And while this method has been successfully used in the detection of rare mutations in mitochondrial DNA samples from normal and cancer tissues [ 24 ], the requirement for a mutation to result in a molecule with an altered melting profile may bias the number of mutations that can be detected. A major discrepancy between our results and those from this earlier report on Pfu fidelity, which may be connected to the differing mutation detection methodologies, can be seen in the mutation spectra results in Table 3.
Because the types of mutations we observe are consistent with previously reported mutational spectra for other Family B polymerases, we believe our method has detected polymerase errors in a bias-free fashion.
The other two enzymes included in our study, KOD polymerase and AccuPrime- Taq High Fidelity, have fidelity values intermediate between Taq polymerase and the higher fidelity enzymes.
In neither of those studies was there a report of the molecular changes leading to mutant colonies. The large difference between these two results, which are from the same research group, serves to highlight the difficulties in making comparisons between studies where there are significant methodological differences.
In the present study, we jod that the mutation spectrum for KOD polymerase is similar to the other B-family polymerases Pfu, Pwo, and Phusion assayed here. According to the vendor, AccuPrime- Taq High Fidelity is an enzyme blend that contains Taq polymerase, a processivity-enhancing protein, and a higher fidelity proofreading polymerase from Pyrococcus species GB-D.
The lower ood rate seen with AccuPrime- Taq most likely arises from the GB-D polymerase editing mistakes introduced by Taq polymerase as opposed to enhanced processivity since increased processivity has been shown to have no significant effect on base substitution errors [ 2227 ]. Detailed analysis on the contribution of each enzyme to the overall mutation spectrum is also precluded by the proprietary enzyme formulation used by the vendor.
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In summary, we have used direct DNA sequencing of cloned PCR products to assay polymerase fidelity and evaluate other aspects of enzyme suitability for large-scale cloning projects. Based on minimizing PCR errors, Pfu polymerase, Pwo polymerase, and Phusion all produce acceptably low levels of mutations. Phusion was observed to produce more indel mutations than Pfu or Pwo polymerases, although the total number of mutations was limited.
This type of mutation is particularly problematic for ORF cloning projects and should be taken into account in the process of enzyme selection. Aside from fidelity considerations, amplification efficiency values were significantly higher for Phusion and Pwo compared to Pfualthough further optimization of the PCR reaction for Pfu would likely improve efficiency values.
And finally, since the application space for PCR technology is huge, with cloning representing only a small fraction, enzymes other than those studied here need to be compared and evaluated based on project-specific needs and challenges. All enzymes and reaction buffers were from commercial sources: For reactions with Phusion, the GC buffer was used.
In all cases, reactions included 0. Template for PCR reactions was miniprep plasmid DNA, with each plasmid template containing a unique target sequence of known sequence and size, ranging from 0.
The target insert was cloned in between the att sites of a pDONR vector, allowing the use of a common primer set for all plasmids.