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Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase.

McInerney P, Adams P, Hadi MZ - Mol Biol Int (2014)

Bottom Line: Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase.Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations.For these enzymes, transition mutations predominate, with little bias observed for type of transition.

View Article: PubMed Central - PubMed

Affiliation: Joint BioEnergy Institute, Emeryville, CA, USA ; Sandia National Laboratories, Livermore, CA, USA.

ABSTRACT
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.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. 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 PCR 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 space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.

No MeSH data available.


Representative agarose gel electrophoresis images of products of PCR amplification of 24 unique DNA targets, using six different enzymes. Each lane contains 1/25 of the entire PCR reaction. Expected product sizes range from 1.4 to 1.7 kb in size.
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fig1: Representative agarose gel electrophoresis images of products of PCR amplification of 24 unique DNA targets, using six different enzymes. Each lane contains 1/25 of the entire PCR reaction. Expected product sizes range from 1.4 to 1.7 kb in size.

Mentions: 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 reactions, thus eliminating the need for target-specific optimizations. Purified plasmid DNA was used as template for PCR, and in all cases vendor-recommended buffers were used. We used small amounts of plasmid template (25 pg/rxn), in order to maximize the number of doublings in the PCR reaction, and the size of insert relative to total plasmid size was taken into account to determine the amount of target fragment present in the template. The PCR protocol used 30 cycles of amplification, with an extension time of 2 minutes/cycle for targets ≤2 kb (82 of 94 targets) and 4 minutes/cycle for targets >2 kb (12 of 94 targets). 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 was 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. As reported in Table 2, amplification 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.


Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase.

McInerney P, Adams P, Hadi MZ - Mol Biol Int (2014)

Representative agarose gel electrophoresis images of products of PCR amplification of 24 unique DNA targets, using six different enzymes. Each lane contains 1/25 of the entire PCR reaction. Expected product sizes range from 1.4 to 1.7 kb in size.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4150459&req=5

fig1: Representative agarose gel electrophoresis images of products of PCR amplification of 24 unique DNA targets, using six different enzymes. Each lane contains 1/25 of the entire PCR reaction. Expected product sizes range from 1.4 to 1.7 kb in size.
Mentions: 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 reactions, thus eliminating the need for target-specific optimizations. Purified plasmid DNA was used as template for PCR, and in all cases vendor-recommended buffers were used. We used small amounts of plasmid template (25 pg/rxn), in order to maximize the number of doublings in the PCR reaction, and the size of insert relative to total plasmid size was taken into account to determine the amount of target fragment present in the template. The PCR protocol used 30 cycles of amplification, with an extension time of 2 minutes/cycle for targets ≤2 kb (82 of 94 targets) and 4 minutes/cycle for targets >2 kb (12 of 94 targets). 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 was 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. As reported in Table 2, amplification 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.

Bottom Line: Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase.Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations.For these enzymes, transition mutations predominate, with little bias observed for type of transition.

View Article: PubMed Central - PubMed

Affiliation: Joint BioEnergy Institute, Emeryville, CA, USA ; Sandia National Laboratories, Livermore, CA, USA.

ABSTRACT
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.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. 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 PCR 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 space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.

No MeSH data available.