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

TY - JOUR

T1 - Real time, in-line optical mapping of single molecules of DNA

AU - Esmek, Franziska M.

AU - Erichlandwehr, Tim

AU - Mors, Dennis H.B.

AU - Czech-Sioli, Manja

AU - Therre, Marlin

AU - Günther, Thomas

AU - Grundhoff, Adam

AU - Fischer, Nicole

AU - Fernandez-Cuesta, Irene

N1 - Funding Information: This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 714073 ). F. Esmek acknowledges the funding from Dr. Hans Messer Foundation, Bad Soden/Ts, STEM research and doctoral scholarship program 2020. This work is also partially supported by the Cluster of Excellence ‘Advanced Imaging of Matter’ of the Deutsche Forschungsgemeinschaft (DFG) - EXC 2056 - project ID 390715994 . This work is also partially funded by the Cluster of Excellence ‘The Hamburg Centre for Ultrafast Imaging’ of the Deutsche Forschungsgemeinschaft (DFG) - EXC 1074 - project ID 194651731 . Funding Information: This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 714073). F. Esmek acknowledges the funding from Dr. Hans Messer Foundation, Bad Soden/Ts, STEM research and doctoral scholarship program 2020. This work is also partially supported by the Cluster of Excellence ?Advanced Imaging of Matter? of the Deutsche Forschungsgemeinschaft (DFG) - EXC 2056 - project ID 390715994. This work is also partially funded by the Cluster of Excellence ?The Hamburg Centre for Ultrafast Imaging? of the Deutsche Forschungsgemeinschaft (DFG) - EXC 1074 - project ID 194651731. Publisher Copyright: © 2021 The Authors

PY - 2021/12

Y1 - 2021/12

N2 - 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.

AB - 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.

KW - DNA analysis

KW - DNA optical Mapping

KW - Laser read-out system

KW - Microfluidic devices

KW - Nanochannel

KW - Nanoimprint

UR - http://www.scopus.com/inward/record.url?scp=85118857941&partnerID=8YFLogxK

U2 - 10.1016/j.biosx.2021.100087

DO - 10.1016/j.biosx.2021.100087

M3 - SCORING: Journal article

AN - SCOPUS:85118857941

VL - 9

JO - Biosensors and Bioelectronics: X

JF - Biosensors and Bioelectronics: X

SN - 2590-1370

M1 - 100087

ER -