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Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction
Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814350/ https://www.ncbi.nlm.nih.gov/pubmed/24013564 http://dx.doi.org/10.1093/nar/gkt782 |
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author | Kutyavin, Igor V. |
author_facet | Kutyavin, Igor V. |
author_sort | Kutyavin, Igor V. |
collection | PubMed |
description | Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed ‘probe-luring’ sequence at their 5′ ends. Hybridization of this sequence to a complementary ‘anchoring’ tail introduced at the 3′ end of a fluorescent probe enables the probe to bind to its target during PCR, and the subsequent probe cleavage results in the florescence signal. As it has been shown in the study, this amplicon-endorsed and guided formation of the probe-target duplex allows the use of extremely short oligonucleotide probes, up to tetranucleotides in length. In particular, the short length of the fluorescent probes makes possible the development of a ‘universal’ probe inventory that is relatively small in size but represents all possible sequence variations. The unparalleled cost-effectiveness of the inventory approach is discussed. Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations. |
format | Online Article Text |
id | pubmed-3814350 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-38143502013-11-04 Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction Kutyavin, Igor V. Nucleic Acids Res Methods Online Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed ‘probe-luring’ sequence at their 5′ ends. Hybridization of this sequence to a complementary ‘anchoring’ tail introduced at the 3′ end of a fluorescent probe enables the probe to bind to its target during PCR, and the subsequent probe cleavage results in the florescence signal. As it has been shown in the study, this amplicon-endorsed and guided formation of the probe-target duplex allows the use of extremely short oligonucleotide probes, up to tetranucleotides in length. In particular, the short length of the fluorescent probes makes possible the development of a ‘universal’ probe inventory that is relatively small in size but represents all possible sequence variations. The unparalleled cost-effectiveness of the inventory approach is discussed. Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations. Oxford University Press 2013-11 2013-09-05 /pmc/articles/PMC3814350/ /pubmed/24013564 http://dx.doi.org/10.1093/nar/gkt782 Text en © The Author(s) 2013. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Methods Online Kutyavin, Igor V. Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction |
title | Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction |
title_full | Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction |
title_fullStr | Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction |
title_full_unstemmed | Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction |
title_short | Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction |
title_sort | use of extremely short förster resonance energy transfer probes in real-time polymerase chain reaction |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814350/ https://www.ncbi.nlm.nih.gov/pubmed/24013564 http://dx.doi.org/10.1093/nar/gkt782 |
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