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CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification
False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049689/ https://www.ncbi.nlm.nih.gov/pubmed/31956898 http://dx.doi.org/10.1093/nar/gkaa017 |
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author | Tian, Bo Minero, Gabriel Antonio S Fock, Jeppe Dufva, Martin Hansen, Mikkel Fougt |
author_facet | Tian, Bo Minero, Gabriel Antonio S Fock, Jeppe Dufva, Martin Hansen, Mikkel Fougt |
author_sort | Tian, Bo |
collection | PubMed |
description | False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies. |
format | Online Article Text |
id | pubmed-7049689 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-70496892020-03-10 CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification Tian, Bo Minero, Gabriel Antonio S Fock, Jeppe Dufva, Martin Hansen, Mikkel Fougt Nucleic Acids Res Methods Online False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies. Oxford University Press 2020-03-18 2020-01-20 /pmc/articles/PMC7049689/ /pubmed/31956898 http://dx.doi.org/10.1093/nar/gkaa017 Text en © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. http://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/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Tian, Bo Minero, Gabriel Antonio S Fock, Jeppe Dufva, Martin Hansen, Mikkel Fougt CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification |
title | CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification |
title_full | CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification |
title_fullStr | CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification |
title_full_unstemmed | CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification |
title_short | CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification |
title_sort | crispr-cas12a based internal negative control for nonspecific products of exponential rolling circle amplification |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049689/ https://www.ncbi.nlm.nih.gov/pubmed/31956898 http://dx.doi.org/10.1093/nar/gkaa017 |
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