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A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations

Digital PCR (dPCR) has great potential for assessing gene editing or gene mutation due to its ability to independently inspect each DNA template in parallel. However, current dPCR methods use a fluorescence-labeled probe to detect gene variation events, and their ability to distinguish variated sequ...

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Detalles Bibliográficos
Autores principales: Li, Bo, Liu, Junhao, Huang, Qilai
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10488114/
https://www.ncbi.nlm.nih.gov/pubmed/37686219
http://dx.doi.org/10.3390/ijms241713405
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author Li, Bo
Liu, Junhao
Huang, Qilai
author_facet Li, Bo
Liu, Junhao
Huang, Qilai
author_sort Li, Bo
collection PubMed
description Digital PCR (dPCR) has great potential for assessing gene editing or gene mutation due to its ability to independently inspect each DNA template in parallel. However, current dPCR methods use a fluorescence-labeled probe to detect gene variation events, and their ability to distinguish variated sequences from the wild-type sequence is limited by the probe’s tolerance to mismatch. To address this, we have developed a novel dPCR method that uses a primer instead of a probe to sense gene variation. The enhanced Taq DNA polymerase in the PCR system has a high mismatch sensitivity, which enables our dPCR method to distinguish gene mutations from wild-type sequences. Compared to current dPCR methods, our method shows superior precision in assessing gene editing efficiency and single-base DNA mutation. This presents a promising opportunity to advance gene editing research and rare gene mutation detection.
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spelling pubmed-104881142023-09-09 A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations Li, Bo Liu, Junhao Huang, Qilai Int J Mol Sci Article Digital PCR (dPCR) has great potential for assessing gene editing or gene mutation due to its ability to independently inspect each DNA template in parallel. However, current dPCR methods use a fluorescence-labeled probe to detect gene variation events, and their ability to distinguish variated sequences from the wild-type sequence is limited by the probe’s tolerance to mismatch. To address this, we have developed a novel dPCR method that uses a primer instead of a probe to sense gene variation. The enhanced Taq DNA polymerase in the PCR system has a high mismatch sensitivity, which enables our dPCR method to distinguish gene mutations from wild-type sequences. Compared to current dPCR methods, our method shows superior precision in assessing gene editing efficiency and single-base DNA mutation. This presents a promising opportunity to advance gene editing research and rare gene mutation detection. MDPI 2023-08-29 /pmc/articles/PMC10488114/ /pubmed/37686219 http://dx.doi.org/10.3390/ijms241713405 Text en © 2023 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
Li, Bo
Liu, Junhao
Huang, Qilai
A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations
title A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations
title_full A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations
title_fullStr A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations
title_full_unstemmed A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations
title_short A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations
title_sort digital pcr method based on highly specific taq for detecting gene editing and mutations
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10488114/
https://www.ncbi.nlm.nih.gov/pubmed/37686219
http://dx.doi.org/10.3390/ijms241713405
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