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Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules
Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5416848/ https://www.ncbi.nlm.nih.gov/pubmed/28077562 http://dx.doi.org/10.1093/nar/gkw1324 |
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author | Kühnemund, Malte Hernández-Neuta, Iván Sharif, Mohd Istiaq Cornaglia, Matteo Gijs, Martin A.M. Nilsson, Mats |
author_facet | Kühnemund, Malte Hernández-Neuta, Iván Sharif, Mohd Istiaq Cornaglia, Matteo Gijs, Martin A.M. Nilsson, Mats |
author_sort | Kühnemund, Malte |
collection | PubMed |
description | Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope. |
format | Online Article Text |
id | pubmed-5416848 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-54168482017-05-05 Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules Kühnemund, Malte Hernández-Neuta, Iván Sharif, Mohd Istiaq Cornaglia, Matteo Gijs, Martin A.M. Nilsson, Mats Nucleic Acids Res Methods Online Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope. Oxford University Press 2017-05-05 2017-01-11 /pmc/articles/PMC5416848/ /pubmed/28077562 http://dx.doi.org/10.1093/nar/gkw1324 Text en © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), 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 Kühnemund, Malte Hernández-Neuta, Iván Sharif, Mohd Istiaq Cornaglia, Matteo Gijs, Martin A.M. Nilsson, Mats Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules |
title | Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules |
title_full | Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules |
title_fullStr | Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules |
title_full_unstemmed | Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules |
title_short | Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules |
title_sort | sensitive and inexpensive digital dna analysis by microfluidic enrichment of rolling circle amplified single-molecules |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5416848/ https://www.ncbi.nlm.nih.gov/pubmed/28077562 http://dx.doi.org/10.1093/nar/gkw1324 |
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