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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...

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Autores principales: Gohl, Daryl M, Auch, Benjamin, Certano, Amanda, LeFrançois, Brice, Bouevitch, Anne, Doukhanine, Evgueni, Fragel, Christina, Macklaim, Jean, Hollister, Emily, Garbe, John, Beckman, Kenneth B
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2021
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.
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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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