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Easily-controllable, helper phage-free single-stranded phagemid production system
BACKGROUND: Single-stranded (ss) DNAs are utilized in various molecular biological and biotechnological applications including the construction of double-stranded DNAs with a DNA lesion, and are commonly prepared by using chimeric phage-plasmids (phagemids) plus M13-derived helper phages. However, t...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9667628/ https://www.ncbi.nlm.nih.gov/pubmed/36380394 http://dx.doi.org/10.1186/s41021-022-00254-1 |
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author | Suzuki, Tetsuya Kamiya, Hiroyuki |
author_facet | Suzuki, Tetsuya Kamiya, Hiroyuki |
author_sort | Suzuki, Tetsuya |
collection | PubMed |
description | BACKGROUND: Single-stranded (ss) DNAs are utilized in various molecular biological and biotechnological applications including the construction of double-stranded DNAs with a DNA lesion, and are commonly prepared by using chimeric phage-plasmids (phagemids) plus M13-derived helper phages. However, the yields of ss DNA with these methods are poorly reproducible, and multiple factors must be optimized. RESULTS: In this report, we describe a new arabinose-inducible ss phagemid production method without helper phage infection. The newly exploited DNA derived from VCSM13 expresses the pII protein, which initiates ss DNA synthesis, under the control of the araBAD promoter. In addition, the packaging signal is deleted in the DNA to reduce the contamination of the phage-derived ss DNA. The phagemid DNA of interest, carrying the M13 origin of replication and the packaging signal, was introduced into bacterial cells maintaining the modified VCSM13 DNA as a plasmid, and the ss phagemid DNA production was induced by arabinose. The DNA recovered from the phage particles had less contamination from VCSM13 DNA, as compared to the conventional method. Moreover, we extended the method to purify the ss DNAs by using an anion-exchange column, to avoid the use of hazardous chemicals. CONCLUSION: Using this combination of methods, large quantities of phagemid ss DNAs of interest can be consistently obtained. |
format | Online Article Text |
id | pubmed-9667628 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-96676282022-11-17 Easily-controllable, helper phage-free single-stranded phagemid production system Suzuki, Tetsuya Kamiya, Hiroyuki Genes Environ Research BACKGROUND: Single-stranded (ss) DNAs are utilized in various molecular biological and biotechnological applications including the construction of double-stranded DNAs with a DNA lesion, and are commonly prepared by using chimeric phage-plasmids (phagemids) plus M13-derived helper phages. However, the yields of ss DNA with these methods are poorly reproducible, and multiple factors must be optimized. RESULTS: In this report, we describe a new arabinose-inducible ss phagemid production method without helper phage infection. The newly exploited DNA derived from VCSM13 expresses the pII protein, which initiates ss DNA synthesis, under the control of the araBAD promoter. In addition, the packaging signal is deleted in the DNA to reduce the contamination of the phage-derived ss DNA. The phagemid DNA of interest, carrying the M13 origin of replication and the packaging signal, was introduced into bacterial cells maintaining the modified VCSM13 DNA as a plasmid, and the ss phagemid DNA production was induced by arabinose. The DNA recovered from the phage particles had less contamination from VCSM13 DNA, as compared to the conventional method. Moreover, we extended the method to purify the ss DNAs by using an anion-exchange column, to avoid the use of hazardous chemicals. CONCLUSION: Using this combination of methods, large quantities of phagemid ss DNAs of interest can be consistently obtained. BioMed Central 2022-11-16 /pmc/articles/PMC9667628/ /pubmed/36380394 http://dx.doi.org/10.1186/s41021-022-00254-1 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Suzuki, Tetsuya Kamiya, Hiroyuki Easily-controllable, helper phage-free single-stranded phagemid production system |
title | Easily-controllable, helper phage-free single-stranded phagemid production system |
title_full | Easily-controllable, helper phage-free single-stranded phagemid production system |
title_fullStr | Easily-controllable, helper phage-free single-stranded phagemid production system |
title_full_unstemmed | Easily-controllable, helper phage-free single-stranded phagemid production system |
title_short | Easily-controllable, helper phage-free single-stranded phagemid production system |
title_sort | easily-controllable, helper phage-free single-stranded phagemid production system |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9667628/ https://www.ncbi.nlm.nih.gov/pubmed/36380394 http://dx.doi.org/10.1186/s41021-022-00254-1 |
work_keys_str_mv | AT suzukitetsuya easilycontrollablehelperphagefreesinglestrandedphagemidproductionsystem AT kamiyahiroyuki easilycontrollablehelperphagefreesinglestrandedphagemidproductionsystem |