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A multiplex platform for digital measurement of circular DNA reaction products

Digital PCR provides high sensitivity and unprecedented accuracy in DNA quantification, but current approaches require dedicated instrumentation and have limited opportunities for multiplexing. Here, we present an isothermal platform for digital enumeration of DNA reaction products in multiplex via...

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Autores principales: Björkesten, Johan, Patil, Sourabh, Fredolini, Claudia, Lönn, Peter, Landegren, Ulf
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367203/
https://www.ncbi.nlm.nih.gov/pubmed/32469060
http://dx.doi.org/10.1093/nar/gkaa419
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author Björkesten, Johan
Patil, Sourabh
Fredolini, Claudia
Lönn, Peter
Landegren, Ulf
author_facet Björkesten, Johan
Patil, Sourabh
Fredolini, Claudia
Lönn, Peter
Landegren, Ulf
author_sort Björkesten, Johan
collection PubMed
description Digital PCR provides high sensitivity and unprecedented accuracy in DNA quantification, but current approaches require dedicated instrumentation and have limited opportunities for multiplexing. Here, we present an isothermal platform for digital enumeration of DNA reaction products in multiplex via standard fluorescence microscopy. Circular DNA strands, which may result from a wide range of molecular detection reactions, are captured on streptavidin-coated surfaces via hybridized biotinylated primers, followed by rolling circle amplification (RCA). The addition of 15% polyethylene glycol 4000 during RCA resulted in uniform, easily recorded reaction products. Immobilized DNA circles were visualized as RCA products with 100% efficiency, as determined by droplet digital PCR. We confirmed previous reports about the influence on RCA by sequence composition and size of RCA templates, and we developed an efficient one-step restaining procedure for sequential multiplexing using toehold-triggered DNA strand displacement. Finally, we exemplify applications of this digital readout platform by demonstrating more than three orders of magnitude improved sensitivity by digital measurement of prostate specific antigen (PSA) (detection threshold ∼100 pg/l), compared to a commercial enzyme-linked immunosorbent assay (ELISA) with analogue readout (detection threshold ∼500 ng/l), using the same antibody pair.
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spelling pubmed-73672032020-07-22 A multiplex platform for digital measurement of circular DNA reaction products Björkesten, Johan Patil, Sourabh Fredolini, Claudia Lönn, Peter Landegren, Ulf Nucleic Acids Res Methods Online Digital PCR provides high sensitivity and unprecedented accuracy in DNA quantification, but current approaches require dedicated instrumentation and have limited opportunities for multiplexing. Here, we present an isothermal platform for digital enumeration of DNA reaction products in multiplex via standard fluorescence microscopy. Circular DNA strands, which may result from a wide range of molecular detection reactions, are captured on streptavidin-coated surfaces via hybridized biotinylated primers, followed by rolling circle amplification (RCA). The addition of 15% polyethylene glycol 4000 during RCA resulted in uniform, easily recorded reaction products. Immobilized DNA circles were visualized as RCA products with 100% efficiency, as determined by droplet digital PCR. We confirmed previous reports about the influence on RCA by sequence composition and size of RCA templates, and we developed an efficient one-step restaining procedure for sequential multiplexing using toehold-triggered DNA strand displacement. Finally, we exemplify applications of this digital readout platform by demonstrating more than three orders of magnitude improved sensitivity by digital measurement of prostate specific antigen (PSA) (detection threshold ∼100 pg/l), compared to a commercial enzyme-linked immunosorbent assay (ELISA) with analogue readout (detection threshold ∼500 ng/l), using the same antibody pair. Oxford University Press 2020-07-27 2020-05-29 /pmc/articles/PMC7367203/ /pubmed/32469060 http://dx.doi.org/10.1093/nar/gkaa419 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
Björkesten, Johan
Patil, Sourabh
Fredolini, Claudia
Lönn, Peter
Landegren, Ulf
A multiplex platform for digital measurement of circular DNA reaction products
title A multiplex platform for digital measurement of circular DNA reaction products
title_full A multiplex platform for digital measurement of circular DNA reaction products
title_fullStr A multiplex platform for digital measurement of circular DNA reaction products
title_full_unstemmed A multiplex platform for digital measurement of circular DNA reaction products
title_short A multiplex platform for digital measurement of circular DNA reaction products
title_sort multiplex platform for digital measurement of circular dna reaction products
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367203/
https://www.ncbi.nlm.nih.gov/pubmed/32469060
http://dx.doi.org/10.1093/nar/gkaa419
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