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Chimeric DNA byproducts in strand displacement amplification using the T7 replisome
Recent advances in next generation sequencing technologies enable reading DNA molecules hundreds of kilobases in length and motivate development of DNA amplification methods capable of producing long amplicons. In vivo, DNA replication is performed not by a single polymerase enzyme, but multiprotein...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9484634/ https://www.ncbi.nlm.nih.gov/pubmed/36121810 http://dx.doi.org/10.1371/journal.pone.0273979 |
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author | Nye, Dillon B. Tanner, Nathan A. |
author_facet | Nye, Dillon B. Tanner, Nathan A. |
author_sort | Nye, Dillon B. |
collection | PubMed |
description | Recent advances in next generation sequencing technologies enable reading DNA molecules hundreds of kilobases in length and motivate development of DNA amplification methods capable of producing long amplicons. In vivo, DNA replication is performed not by a single polymerase enzyme, but multiprotein complexes called replisomes. Here, we investigate strand-displacement amplification reactions using the T7 replisome, a macromolecular complex of a helicase, a single-stranded DNA binding protein, and a DNA polymerase. The T7 replisome may initiate processive DNA synthesis from DNA nicks, and the reaction of a 48 kilobase linear double stranded DNA substrate with the T7 replisome and nicking endonucleases is shown to produce discrete DNA amplicons. To gain a mechanistic understanding of this reaction, we utilized Oxford Nanopore long-read sequencing technology. Sequence analysis of the amplicons revealed chimeric DNA reads and uncovered a connection between template switching and polymerase exonuclease activity. Nanopore sequencing provides insight to guide the further development of isothermal amplification methods for long DNA, and our results highlight the need for high-specificity, high-turnover nicking endonucleases to initiate DNA amplification without thermal denaturation. |
format | Online Article Text |
id | pubmed-9484634 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-94846342022-09-20 Chimeric DNA byproducts in strand displacement amplification using the T7 replisome Nye, Dillon B. Tanner, Nathan A. PLoS One Research Article Recent advances in next generation sequencing technologies enable reading DNA molecules hundreds of kilobases in length and motivate development of DNA amplification methods capable of producing long amplicons. In vivo, DNA replication is performed not by a single polymerase enzyme, but multiprotein complexes called replisomes. Here, we investigate strand-displacement amplification reactions using the T7 replisome, a macromolecular complex of a helicase, a single-stranded DNA binding protein, and a DNA polymerase. The T7 replisome may initiate processive DNA synthesis from DNA nicks, and the reaction of a 48 kilobase linear double stranded DNA substrate with the T7 replisome and nicking endonucleases is shown to produce discrete DNA amplicons. To gain a mechanistic understanding of this reaction, we utilized Oxford Nanopore long-read sequencing technology. Sequence analysis of the amplicons revealed chimeric DNA reads and uncovered a connection between template switching and polymerase exonuclease activity. Nanopore sequencing provides insight to guide the further development of isothermal amplification methods for long DNA, and our results highlight the need for high-specificity, high-turnover nicking endonucleases to initiate DNA amplification without thermal denaturation. Public Library of Science 2022-09-19 /pmc/articles/PMC9484634/ /pubmed/36121810 http://dx.doi.org/10.1371/journal.pone.0273979 Text en © 2022 Nye, Tanner https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Nye, Dillon B. Tanner, Nathan A. Chimeric DNA byproducts in strand displacement amplification using the T7 replisome |
title | Chimeric DNA byproducts in strand displacement amplification using the T7 replisome |
title_full | Chimeric DNA byproducts in strand displacement amplification using the T7 replisome |
title_fullStr | Chimeric DNA byproducts in strand displacement amplification using the T7 replisome |
title_full_unstemmed | Chimeric DNA byproducts in strand displacement amplification using the T7 replisome |
title_short | Chimeric DNA byproducts in strand displacement amplification using the T7 replisome |
title_sort | chimeric dna byproducts in strand displacement amplification using the t7 replisome |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9484634/ https://www.ncbi.nlm.nih.gov/pubmed/36121810 http://dx.doi.org/10.1371/journal.pone.0273979 |
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