Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase
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 numero...
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| Format: | Online |
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Hindawi Publishing Corporation
2014
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4150459/ |
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pubmed-4150459 |
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pubmed-41504592014-09-07 Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase McInerney, Peter Adams, Paul Hadi, Masood Z. Research Article 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. Hindawi Publishing Corporation 2014 2014-08-17 /pmc/articles/PMC4150459/ /pubmed/25197572 http://dx.doi.org/10.1155/2014/287430 Text en Copyright © 2014 Peter McInerney et al. https://creativecommons.org/licenses/by/3.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
McInerney, Peter Adams, Paul Hadi, Masood Z. |
| spellingShingle |
McInerney, Peter Adams, Paul Hadi, Masood Z. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase |
| author_facet |
McInerney, Peter Adams, Paul Hadi, Masood Z. |
| author_sort |
McInerney, Peter |
| title |
Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase |
| title_short |
Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase |
| title_full |
Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase |
| title_fullStr |
Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase |
| title_full_unstemmed |
Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase |
| title_sort |
error rate comparison during polymerase chain reaction by dna polymerase |
| description |
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. |
| publisher |
Hindawi Publishing Corporation |
| publishDate |
2014 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4150459/ |
| _version_ |
1613129729513095168 |