Real time, in-line optical mapping of single molecules of DNA

DNA optical mapping in nanochannels allows studying intact molecules and analyzing their long-range structure at the single-molecule level. Recent efforts have demonstrated that optical mapping can be used for various biomedical applications, such as bacteria identification, analysis of tumor cells,...

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Autores principales:	Franziska M. Esmek, Tim Erichlandwehr, Dennis H.B. Mors, Manja Czech-Sioli, Marlin Therre, Thomas Günther, Adam Grundhoff, Nicole Fischer, Irene Fernandez-Cuesta
Formato:	article
Lenguaje:	EN
Publicado:	Elsevier 2021
Materias:	Microfluidic devices Nanoimprint Nanochannel DNA optical Mapping Laser read-out system DNA analysis Biotechnology TP248.13-248.65
Acceso en línea:	https://doaj.org/article/88d8f1f8367647288a758ce1b80d4f29
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spelling	oai:doaj.org-article:88d8f1f8367647288a758ce1b80d4f292021-11-12T04:46:51ZReal time, in-line optical mapping of single molecules of DNA2590-137010.1016/j.biosx.2021.100087https://doaj.org/article/88d8f1f8367647288a758ce1b80d4f292021-12-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S2590137021000236https://doaj.org/toc/2590-1370DNA optical mapping in nanochannels allows studying intact molecules and analyzing their long-range structure at the single-molecule level. Recent efforts have demonstrated that optical mapping can be used for various biomedical applications, such as bacteria identification, analysis of tumor cells, or whole-genome mapping. However, techniques for optical mapping are still slow and restricted to specialized labs. Here, we show a complete methodology for real-time DNA optical mapping on-chip, which is simple and offers high throughput. It does not require a microscope nor a high sensitivity camera to read the barcode, nor the use of external forces (like electrophoresis) to drive the molecules into the nanochannels. The DNA molecules are labelled with different methods, which allows barcoding known and unknown molecules. The barcoded DNA sample is analyzed in single-use, plastic nanoimprinted fluidic devices, which are versatile platforms to manipulate and stretch the molecules in nanochannels. And the fluorescent signal of the molecules is recorded in-line, in real time, with a laser system and a photon counter. With this methodology, we obtained barcodes of molecules with periodic sequences, where we marked one site per period. Furthermore, we barcoded the DNA of bacteriophages (Lambda and T4) and of a tumor virus (the Kaposi Sarcoma Herpesvirus, KSHV) by competitive binding, and obtained their unique fingerprints. Interestingly, this method succeeds in the correct detection (length and number) of highly repetitive structures such as the terminal repeat region of KSHV. These results show the versatility of the proposed methodology for fast (few milliseconds per molecule), low cost, high throughput (tens of molecules per minute) DNA analysis on-demand for biomedical applications. In particular, it can be used to analyze DNA with repeated sequences complementing other commercial techniques.Franziska M. EsmekTim ErichlandwehrDennis H.B. MorsManja Czech-SioliMarlin TherreThomas GüntherAdam GrundhoffNicole FischerIrene Fernandez-CuestaElsevierarticleMicrofluidic devicesNanoimprintNanochannelDNA optical MappingLaser read-out systemDNA analysisBiotechnologyTP248.13-248.65ENBiosensors and Bioelectronics: X, Vol 9, Iss , Pp 100087- (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microfluidic devices Nanoimprint Nanochannel DNA optical Mapping Laser read-out system DNA analysis Biotechnology TP248.13-248.65
spellingShingle	Microfluidic devices Nanoimprint Nanochannel DNA optical Mapping Laser read-out system DNA analysis Biotechnology TP248.13-248.65 Franziska M. Esmek Tim Erichlandwehr Dennis H.B. Mors Manja Czech-Sioli Marlin Therre Thomas Günther Adam Grundhoff Nicole Fischer Irene Fernandez-Cuesta Real time, in-line optical mapping of single molecules of DNA
description	DNA optical mapping in nanochannels allows studying intact molecules and analyzing their long-range structure at the single-molecule level. Recent efforts have demonstrated that optical mapping can be used for various biomedical applications, such as bacteria identification, analysis of tumor cells, or whole-genome mapping. However, techniques for optical mapping are still slow and restricted to specialized labs. Here, we show a complete methodology for real-time DNA optical mapping on-chip, which is simple and offers high throughput. It does not require a microscope nor a high sensitivity camera to read the barcode, nor the use of external forces (like electrophoresis) to drive the molecules into the nanochannels. The DNA molecules are labelled with different methods, which allows barcoding known and unknown molecules. The barcoded DNA sample is analyzed in single-use, plastic nanoimprinted fluidic devices, which are versatile platforms to manipulate and stretch the molecules in nanochannels. And the fluorescent signal of the molecules is recorded in-line, in real time, with a laser system and a photon counter. With this methodology, we obtained barcodes of molecules with periodic sequences, where we marked one site per period. Furthermore, we barcoded the DNA of bacteriophages (Lambda and T4) and of a tumor virus (the Kaposi Sarcoma Herpesvirus, KSHV) by competitive binding, and obtained their unique fingerprints. Interestingly, this method succeeds in the correct detection (length and number) of highly repetitive structures such as the terminal repeat region of KSHV. These results show the versatility of the proposed methodology for fast (few milliseconds per molecule), low cost, high throughput (tens of molecules per minute) DNA analysis on-demand for biomedical applications. In particular, it can be used to analyze DNA with repeated sequences complementing other commercial techniques.
format	article
author	Franziska M. Esmek Tim Erichlandwehr Dennis H.B. Mors Manja Czech-Sioli Marlin Therre Thomas Günther Adam Grundhoff Nicole Fischer Irene Fernandez-Cuesta
author_facet	Franziska M. Esmek Tim Erichlandwehr Dennis H.B. Mors Manja Czech-Sioli Marlin Therre Thomas Günther Adam Grundhoff Nicole Fischer Irene Fernandez-Cuesta
author_sort	Franziska M. Esmek
title	Real time, in-line optical mapping of single molecules of DNA
title_short	Real time, in-line optical mapping of single molecules of DNA
title_full	Real time, in-line optical mapping of single molecules of DNA
title_fullStr	Real time, in-line optical mapping of single molecules of DNA
title_full_unstemmed	Real time, in-line optical mapping of single molecules of DNA
title_sort	real time, in-line optical mapping of single molecules of dna
publisher	Elsevier
publishDate	2021
url	https://doaj.org/article/88d8f1f8367647288a758ce1b80d4f29
work_keys_str_mv	AT franziskamesmek realtimeinlineopticalmappingofsinglemoleculesofdna AT timerichlandwehr realtimeinlineopticalmappingofsinglemoleculesofdna AT dennishbmors realtimeinlineopticalmappingofsinglemoleculesofdna AT manjaczechsioli realtimeinlineopticalmappingofsinglemoleculesofdna AT marlintherre realtimeinlineopticalmappingofsinglemoleculesofdna AT thomasgunther realtimeinlineopticalmappingofsinglemoleculesofdna AT adamgrundhoff realtimeinlineopticalmappingofsinglemoleculesofdna AT nicolefischer realtimeinlineopticalmappingofsinglemoleculesofdna AT irenefernandezcuesta realtimeinlineopticalmappingofsinglemoleculesofdna
_version_	1718431240968732672

Real time, in-line optical mapping of single molecules of DNA

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