Self-Assembly of a Bio-Driven Single Molecule DNA Sequencer

DNA sequencing reads the order in which the four nucleotide bases – adenine (A), thymine (T), cytosine (C) and guanine (G) – arrange into the long biopolymer that encodes the genes of all living organisms. The ability to sequence DNA has revolutionised the biological sciences and is having a rapidly expanding impact on multiple industries including medicine, agriculture, and biotechnology. The DNA sequencing market is predicted to reach $60 billion by 2030. Nonetheless, there exist serious challenges for the current wave of industry-leading high-throughput sequencing methods including the need for large quantities of DNA, limitations on the maximum DNA length that can be continuously read, and the computational resources needed to build the sequence from raw data. A highly coveted goal is to accurately read the sequence of a single DNA molecule in real time. Recently, significant progress has been made with single-molecule sequencing technologies and two emerging technologies – Oxford Nanopore’s ‘current blockade’ and PacBio’s ‘zero mode waveguide’ sequencing – now lead the market. Both technologies use methods subject to fundamental limits in our ability to precisely manipulate a single DNA molecule and confine the observation volume to a single nucleotide. These limitations result in substantial error rates, and thus accurate sequencing still requires multiple repeated sequencing reactions to attain accuracy statistically. True single-molecule DNA sequencing remains an unsolved problem. Here, we describe a new method for single-molecule DNA sequencing, by enhancing the fluorescent signal from single DNA bases as they are incorporated in the correct sequence by the enzyme DNA polymerase. Fluorescence enhancement is achieved by the use of a plasmonic nanoantenna. Chapter 1 provides an overview of existing state of the art technologies and underlying theory behind plasmonic nanoantenna sequencing. Chapter 2 provides the materials and methods. Chapter 3 describes the embodiment of the plasmonic nanoantenna DNA sequencer. Chapter 4 characterises the fluorescence enhancement of the plasmonic nanoantenna. Chapter 5 provides proof of concept experiments with examples of reactions that yield correct DNA sequences. This method has accurately identified mostly the first 12 coloured bases compared to the expected 27-color sequence in the alignment, which holds great promise for advancing the field of DNA sequencing to achieve sequencing of individual DNA molecules.

Grantor dc:publisher

UNSW, Sydney

Year dc:date

2024

Author dc:creator

• Huang, Min ; https://orcid.org/0000-0003-4538-5787

• DNA sequencing
• plasmonic nanoantenna
• single-molecule DNA sequencing
• fluorescence enhancement
• anzsrc-for: 310607 Nanobiotechnology

Statement dc:rights

• open access
• CC BY 4.0
• free_to_read

Licence

• https://purl.org/coar/access_right/c_abf2
• https://creativecommons.org/licenses/by/4.0/

Language dc:language

en

Identifier

https://doi.org/10.26190/unsworks/25550

OAI identifier oai:identifier

oai:unsworks.library.unsw.edu.au:1959.4/101847