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Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts
Restriction endonucleases are expressed in all bacteria investigated so far and play an essential role for the bacterial defense against viral infections. Besides their important biological role, restriction endonucleases are of great use for different biotechnological purposes and are indispensable...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9608553/ https://www.ncbi.nlm.nih.gov/pubmed/36298113 http://dx.doi.org/10.3390/s22207763 |
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author | Petersen, Kamilla Vandsø Tesauro, Cinzia Hede, Marianne Smedegaard Pages, Camilla Marcussen, Lærke Bay Keller, Josephine Geertsen Bugge, Magnus Holm, Kasper Bjergbæk, Lotte Stougaard, Magnus Wejse, Christian Knudsen, Birgitta R. |
author_facet | Petersen, Kamilla Vandsø Tesauro, Cinzia Hede, Marianne Smedegaard Pages, Camilla Marcussen, Lærke Bay Keller, Josephine Geertsen Bugge, Magnus Holm, Kasper Bjergbæk, Lotte Stougaard, Magnus Wejse, Christian Knudsen, Birgitta R. |
author_sort | Petersen, Kamilla Vandsø |
collection | PubMed |
description | Restriction endonucleases are expressed in all bacteria investigated so far and play an essential role for the bacterial defense against viral infections. Besides their important biological role, restriction endonucleases are of great use for different biotechnological purposes and are indispensable for many cloning and sequencing procedures. Methods for specific detection of restriction endonuclease activities can therefore find broad use for many purposes. In the current study, we demonstrate proof-of-concept for a new principle for the detection of restriction endonuclease activities. The method is based on rolling circle amplification of circular DNA products that can only be formed upon restriction digestion of specially designed DNA substrates. By combining the activity of the target restriction endonuclease with the highly specific Cre recombinase to generate DNA circles, we demonstrate specific detection of selected restriction endonuclease activities even in crude cell extracts. This is, to our knowledge, the first example of a sensor system that allows activity measurements of restriction endonucleases in crude samples. The presented sensor system may prove valuable for future characterization of bacteria species or strains based on their expression of restriction endonucleases as well as for quantification of restriction endonuclease activities directly in extracts from recombinant cells. |
format | Online Article Text |
id | pubmed-9608553 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-96085532022-10-28 Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts Petersen, Kamilla Vandsø Tesauro, Cinzia Hede, Marianne Smedegaard Pages, Camilla Marcussen, Lærke Bay Keller, Josephine Geertsen Bugge, Magnus Holm, Kasper Bjergbæk, Lotte Stougaard, Magnus Wejse, Christian Knudsen, Birgitta R. Sensors (Basel) Article Restriction endonucleases are expressed in all bacteria investigated so far and play an essential role for the bacterial defense against viral infections. Besides their important biological role, restriction endonucleases are of great use for different biotechnological purposes and are indispensable for many cloning and sequencing procedures. Methods for specific detection of restriction endonuclease activities can therefore find broad use for many purposes. In the current study, we demonstrate proof-of-concept for a new principle for the detection of restriction endonuclease activities. The method is based on rolling circle amplification of circular DNA products that can only be formed upon restriction digestion of specially designed DNA substrates. By combining the activity of the target restriction endonuclease with the highly specific Cre recombinase to generate DNA circles, we demonstrate specific detection of selected restriction endonuclease activities even in crude cell extracts. This is, to our knowledge, the first example of a sensor system that allows activity measurements of restriction endonucleases in crude samples. The presented sensor system may prove valuable for future characterization of bacteria species or strains based on their expression of restriction endonucleases as well as for quantification of restriction endonuclease activities directly in extracts from recombinant cells. MDPI 2022-10-13 /pmc/articles/PMC9608553/ /pubmed/36298113 http://dx.doi.org/10.3390/s22207763 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Petersen, Kamilla Vandsø Tesauro, Cinzia Hede, Marianne Smedegaard Pages, Camilla Marcussen, Lærke Bay Keller, Josephine Geertsen Bugge, Magnus Holm, Kasper Bjergbæk, Lotte Stougaard, Magnus Wejse, Christian Knudsen, Birgitta R. Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts |
title | Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts |
title_full | Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts |
title_fullStr | Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts |
title_full_unstemmed | Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts |
title_short | Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts |
title_sort | rolling circle enhanced detection of specific restriction endonuclease activities in crude cell extracts |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9608553/ https://www.ncbi.nlm.nih.gov/pubmed/36298113 http://dx.doi.org/10.3390/s22207763 |
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