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Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification

Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism dete...

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Detalles Bibliográficos
Autores principales: Yi, Jizu, Zhang, Wandi, Zhang, David Y.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2006
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1488881/
https://www.ncbi.nlm.nih.gov/pubmed/16822854
http://dx.doi.org/10.1093/nar/gkl261
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author Yi, Jizu
Zhang, Wandi
Zhang, David Y.
author_facet Yi, Jizu
Zhang, Wandi
Zhang, David Y.
author_sort Yi, Jizu
collection PubMed
description Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R(2) = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays.
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spelling pubmed-14888812006-07-25 Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification Yi, Jizu Zhang, Wandi Zhang, David Y. Nucleic Acids Res Methods Online Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R(2) = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays. Oxford University Press 2006 2006-07-05 /pmc/articles/PMC1488881/ /pubmed/16822854 http://dx.doi.org/10.1093/nar/gkl261 Text en © 2006 The Author(s)
spellingShingle Methods Online
Yi, Jizu
Zhang, Wandi
Zhang, David Y.
Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification
title Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification
title_full Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification
title_fullStr Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification
title_full_unstemmed Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification
title_short Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification
title_sort molecular zipper: a fluorescent probe for real-time isothermal dna amplification
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1488881/
https://www.ncbi.nlm.nih.gov/pubmed/16822854
http://dx.doi.org/10.1093/nar/gkl261
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