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Dissecting and tuning primer editing by proofreading polymerases
Proofreading polymerases have 3′ to 5′ exonuclease activity that allows the excision and correction of mis-incorporated bases during DNA replication. In a previous study, we demonstrated that in addition to correcting substitution errors and lowering the error rate of DNA amplification, proofreading...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8421222/ https://www.ncbi.nlm.nih.gov/pubmed/34125893 http://dx.doi.org/10.1093/nar/gkab471 |
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author | Gohl, Daryl M Auch, Benjamin Certano, Amanda LeFrançois, Brice Bouevitch, Anne Doukhanine, Evgueni Fragel, Christina Macklaim, Jean Hollister, Emily Garbe, John Beckman, Kenneth B |
author_facet | Gohl, Daryl M Auch, Benjamin Certano, Amanda LeFrançois, Brice Bouevitch, Anne Doukhanine, Evgueni Fragel, Christina Macklaim, Jean Hollister, Emily Garbe, John Beckman, Kenneth B |
author_sort | Gohl, Daryl M |
collection | PubMed |
description | Proofreading polymerases have 3′ to 5′ exonuclease activity that allows the excision and correction of mis-incorporated bases during DNA replication. In a previous study, we demonstrated that in addition to correcting substitution errors and lowering the error rate of DNA amplification, proofreading polymerases can also edit PCR primers to match template sequences. Primer editing is a feature that can be advantageous in certain experimental contexts, such as amplicon-based microbiome profiling. Here we develop a set of synthetic DNA standards to report on primer editing activity and use these standards to dissect this phenomenon. The primer editing standards allow next-generation sequencing-based enzymological measurements, reveal the extent of editing, and allow the comparison of different polymerases and cycling conditions. We demonstrate that proofreading polymerases edit PCR primers in a concentration-dependent manner, and we examine whether primer editing exhibits any sequence specificity. In addition, we use these standards to show that primer editing is tunable through the incorporation of phosphorothioate linkages. Finally, we demonstrate the ability of primer editing to robustly rescue the drop-out of taxa with 16S rRNA gene-targeting primer mismatches using mock communities and human skin microbiome samples. |
format | Online Article Text |
id | pubmed-8421222 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-84212222021-09-09 Dissecting and tuning primer editing by proofreading polymerases Gohl, Daryl M Auch, Benjamin Certano, Amanda LeFrançois, Brice Bouevitch, Anne Doukhanine, Evgueni Fragel, Christina Macklaim, Jean Hollister, Emily Garbe, John Beckman, Kenneth B Nucleic Acids Res Methods Online Proofreading polymerases have 3′ to 5′ exonuclease activity that allows the excision and correction of mis-incorporated bases during DNA replication. In a previous study, we demonstrated that in addition to correcting substitution errors and lowering the error rate of DNA amplification, proofreading polymerases can also edit PCR primers to match template sequences. Primer editing is a feature that can be advantageous in certain experimental contexts, such as amplicon-based microbiome profiling. Here we develop a set of synthetic DNA standards to report on primer editing activity and use these standards to dissect this phenomenon. The primer editing standards allow next-generation sequencing-based enzymological measurements, reveal the extent of editing, and allow the comparison of different polymerases and cycling conditions. We demonstrate that proofreading polymerases edit PCR primers in a concentration-dependent manner, and we examine whether primer editing exhibits any sequence specificity. In addition, we use these standards to show that primer editing is tunable through the incorporation of phosphorothioate linkages. Finally, we demonstrate the ability of primer editing to robustly rescue the drop-out of taxa with 16S rRNA gene-targeting primer mismatches using mock communities and human skin microbiome samples. Oxford University Press 2021-06-14 /pmc/articles/PMC8421222/ /pubmed/34125893 http://dx.doi.org/10.1093/nar/gkab471 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Gohl, Daryl M Auch, Benjamin Certano, Amanda LeFrançois, Brice Bouevitch, Anne Doukhanine, Evgueni Fragel, Christina Macklaim, Jean Hollister, Emily Garbe, John Beckman, Kenneth B Dissecting and tuning primer editing by proofreading polymerases |
title | Dissecting and tuning primer editing by proofreading polymerases |
title_full | Dissecting and tuning primer editing by proofreading polymerases |
title_fullStr | Dissecting and tuning primer editing by proofreading polymerases |
title_full_unstemmed | Dissecting and tuning primer editing by proofreading polymerases |
title_short | Dissecting and tuning primer editing by proofreading polymerases |
title_sort | dissecting and tuning primer editing by proofreading polymerases |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8421222/ https://www.ncbi.nlm.nih.gov/pubmed/34125893 http://dx.doi.org/10.1093/nar/gkab471 |
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