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, 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. © 2021 The Authors