Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Tian, Bo, Minero, Gabriel Antonio S, Fock, Jeppe, Dufva, Martin, Hansen, Mikkel Fougt
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
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
_version_ 1783502491580628992
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
work_keys_str_mv AT tianbo crisprcas12abasedinternalnegativecontrolfornonspecificproductsofexponentialrollingcircleamplification
AT minerogabrielantonios crisprcas12abasedinternalnegativecontrolfornonspecificproductsofexponentialrollingcircleamplification
AT fockjeppe crisprcas12abasedinternalnegativecontrolfornonspecificproductsofexponentialrollingcircleamplification
AT dufvamartin crisprcas12abasedinternalnegativecontrolfornonspecificproductsofexponentialrollingcircleamplification
AT hansenmikkelfougt crisprcas12abasedinternalnegativecontrolfornonspecificproductsofexponentialrollingcircleamplification