Single molecule DNA sequencing in gold.

Lead Research Organisation: University of Southampton
Department Name: Optoelectronics Research Centre

Abstract

The human genome contains 3164.7 million nucleic acid bases (adenine, guanine, cytosine, thymine) and it is estimated that the length of all the DNA strands in a single cell (if all the strands were placed end to end) is about two metres. The human genome sequence was completed at the start of the millennium; this resulted in significant public and scientific interest in understanding the DNA sequence 'code' and how it is 'translated'. The sequence of the genome provides information about our ancestry, hereditary diseases, our features (such as eye, skin or hair colour) and our physiological 'make-up'. In order to truly understand the genome sequence it would be desirable to have simpler DNA sequencing methods so that many more genomes could be sequenced. Currently, methods require expensive reagents, are laborious and take a long time. Despite the fact that the human genome was sequenced a decade ago and better DNA sequencing methods have been since developed, a simple, cheap, fast, accurate DNA sequencing method is still an important goal. In our view, what is needed is a small scale technology, something that works like a hard disk drive, where a tiny read head is scanned past the stored information (the DNA strand) and the information (the sequence) is read directly without need for any complex processing of the genomic DNA molecule. We propose to flow DNA molecules through a nanopore that will act as a 'read head' and detect and identify the nucleic acids in the DNA sequence by their response to laser excitation. Identifying the DNA base sequence in this way will be highly ac curate, and capable of detecting damage or modification to particular bases from environmental or cellular processes which can control the switching on and off of genes. DNA sequencing techniques are crucial to obtain a better understand of all organisms, not just humans, and a fast, cheap DNA sequencing method will be able to answer many more questions as well as also provide a diagnostic tool. These studies will provide proof of concept data appropriate to demonstrate a very new DNA sequencing approach.

Technical Summary

A one year proof of concept study is proposed to demonstrate a new method for single molecule DNA sequencing that allows for the direct spectral identification of the nucleic acid bases in the sequence. This approach should ultimately allow for fast, accurate sequencing without the need for enzymes such as polymerase. Nanostructures will be fabricated and DNA molecules will be transported in fluid through the structures. An optical set-up will be built that allows for the direct detection of DNA nucleic acid bases in a sequence dependent manner. This study will build upon our expertise in DNA chemistry, bioanalytical devices and near-field optical approaches.

Planned Impact

The UK has a significant and growing commercial activity in the field of diagnostics and DNA sequencing technologies. A methodology that provides improvements over existing DNA sequencing methods has a clear commercial potential. It is important to be realistic - a one year 'proof of concept' study is proposed and this is not going to reach a technology readiness level that is anywhere close to a product that could be commercialised or that could be of interest to venture capitalists. Even so, we have had informal discussions with Renishaw Diagnostics, who have shown a strong interest and we plan to cement a more solid collaboration in the longer-term with them. In addition members within the collaborative team have (i) a track-record for commercialising their research through start-up companies and (ii) current collaborative activities with members of commercial companies involved in DNA analysis and sequencing technology (as outlined in our 'Pathways to Impact' document). Nanoporous technologies, of the type proposed, might also be useful for the detection other small biomolecules such as metabolic products (in body fluids), pharmaceutical agents and environmental pollutants. In order to achieve the impact of our research we will engage with members of the environmental and pharmaceutical, industrial, public or private sectors, once our results are protected. The realistic timescales for the benefits to be achieved will be beyond that the 'proof of concept' study proposed here. The research is clearly 'high risk/high reward' and very adventurous (thus fitting the 'Early concept, exploratory investigations of new tools, technologies and resources' within the current call). We envisage that a further 3 year period of study is required to demonstrate the methodology for 'real' DNA sequencing in a robust and reliable manner. The impact of achieving this would be substantial.
 
Description i) Fabrication of three-dimensional gold nanoporous structure with optimal geometries for DNA sequencing applications. Two different three-dimensional gold nanoporous structures. The silicon Klarite samples were provided by Renishaw Diagnoitics and used to produce 100nm thick, three-dimensional gold films. Subsequent milling using a helium ion microscope provided a 50nm hole. The structure and hole size was chosen based upon theoretical studies performed in a new collaboration with Dr Martin Charlton and his PhD student, Swe Zin. These results were disseminated by the post-doctoral researcher appointed to the grant in Dr Melvin's lab.

ii) Direct detection of the nucleic acid bases by SERS approaches using the 3D gold structures. Substrate fabrication was optimised to provide direct detection by SERS of the nucleic acid bases within DNA sequences, this detection limit had only previously been observed for nanoparticles.
Exploitation Route Potential use in DNA sequencing (currently extending this programme with additional funding achieved)
Sectors Electronics,Pharmaceuticals and Medical Biotechnology

 
Description Shedding light on DNA sequence information
Amount £9,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2014 
End 06/2014
 
Title Fabrication of 3D structure gold films with thickness of the nanometer scale with precise geometries and high smoothness for DNA sequencing applications 
Description Based upon theoretical designs 3D gold films of specific geometries and high smoothness were fabricated. The method involved a templated method whereby a thin Teflon coating with a subsequent gold film evaporation provided a film which conformed to the arrays of pyramid structured surface. Following lift-off of the film, nanopores were milled into the structures at locations for DNA translocation. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact First published example of method based upon micro and nanoengineering of 3D structure film of designed geometries. This has only just been disseminated, but attracting interest of other research groups. 
 
Title Theoretical model of electric field enhancement in nanopores within 3D structures 
Description DiffractMOD employs the Rigorous Coupled Wave Analysis method to obtain the backward diffraction efficiency of the surface when illuminated by a plane wave from above (normal to the upper face of the pyramid cavities). A transmission line analysis is applied to an expansion of the refractive index and Electromagnetic field vectors in Fourier space on a cell-by-cell basis, across a fine mesh superimposed on the three-dimensional representation of the refractive index. In our simulation, periodic boundary conditions are used at the sides and number of harmonics is set to '7' in x and y coordinate transverse to the primary direction, z. The number of harmonics is used to expand the refractive index and field in Fourier space resulting in accurate simulation. Simulations are performed for a wide spectral range (400 nm to 1500 nm with 5 nm steps), typically with TM-polarisation. The designs were optimised to provide electric field enhancement within the nanopore to facilitate fluorescence 'read-out' of fluorophores as they transit the pore. 
Type Of Material Computer model/algorithm 
Year Produced 2015 
Provided To Others? Yes  
Impact This information was fully disclosed in the supplementary information of two papers published in 2015 and 2016. In addition the data was provided in movie format to facilitate full disclosure of the data. 
URL http://iopscience.iop.org/0957-4484/27/6/065302/media/video1.mp4
 
Description Cafe Scientifique 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Cafe Scientifique at the Isle of Wright. There are significant problems for residents on the island to get any sort of opportunity to attend any sort of public dissemination activity associated with science. It has always been disappointing to me that the educational results on the island are lower than on the mainland of Hampshire. A trip to the Isle of Wright from my department in the University is challenging and required approximately 3 hrs of travel one-way. Thus although this was a time-consuming activity the audience was especially enthusiastic. It was clear that the organisers struggled to attract speakers. The presentation that I delivered was appropriate to an audience with a very limited knowledge of genetics (and epigenetics) and the application of sequencing for stratified medicine. This resulted in a discussion of 1.5 hrs and only stopped because it was necessary to get a boat back to Southampton.
Year(s) Of Engagement Activity 2017
 
Description Science Cafe, at local pub 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Presentation and questions and Answer session: The different ways DNA can

be used for medicine and diagnostics, including super fast DNA testing.

23 people, 1.5 hours interspersed with Q&A Presentation suitable for pub

request to participate in further dissemination activities to public
Year(s) Of Engagement Activity 2012