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A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells
Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryo...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917819/ https://www.ncbi.nlm.nih.gov/pubmed/27334801 http://dx.doi.org/10.1038/srep28000 |
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author | Soejima, Takashi Xiao, Jin-zhong Abe, Fumiaki |
author_facet | Soejima, Takashi Xiao, Jin-zhong Abe, Fumiaki |
author_sort | Soejima, Takashi |
collection | PubMed |
description | Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 10(0) cfu/ml for the test sample compared with a detection limit of 1.6 × 10(3) cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research. |
format | Online Article Text |
id | pubmed-4917819 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-49178192016-06-27 A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells Soejima, Takashi Xiao, Jin-zhong Abe, Fumiaki Sci Rep Article Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 10(0) cfu/ml for the test sample compared with a detection limit of 1.6 × 10(3) cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research. Nature Publishing Group 2016-06-23 /pmc/articles/PMC4917819/ /pubmed/27334801 http://dx.doi.org/10.1038/srep28000 Text en Copyright © 2016, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
spellingShingle | Article Soejima, Takashi Xiao, Jin-zhong Abe, Fumiaki A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells |
title | A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells |
title_full | A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells |
title_fullStr | A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells |
title_full_unstemmed | A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells |
title_short | A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells |
title_sort | novel mechanism for direct real-time polymerase chain reaction that does not require dna isolation from prokaryotic cells |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4917819/ https://www.ncbi.nlm.nih.gov/pubmed/27334801 http://dx.doi.org/10.1038/srep28000 |
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