DNA Sequencing

The need for label-free, single molecule sequencing

Many existing DNA sequencing technologies are burdened by the need to use fluorescent molecular labels and optical hardware to differentiate the types of DNA base. This process is expensive, due to the cost of reagents and sophisticated instrumentation that is needed to detect and interpret the photon signal into DNA sequence data. The workflow and data management can also be complex with heavy emphasis on skilled labour.  

Most sequencing methods also require amplification of the DNA before sequencing. This process also adds time and expense and can result in amplification errors, leading to inaccurate sequence data.

Nanopores offer a label-free, electrical, single-molecule DNA sequencing method, obviating the need for amplification or labelling by detecting a direct electrical signal. Whilst the evolution of other technologies relies on improvements in existing chemical, optical or bioinformatics procedures, nanopores will bypass these to deliver a genuinely revolutionary sequencing method. 

Exonuclease sequencing: Oxford Nanopore's first generation of sequencing technology

The natural α-hemolysin nanopore alone is not capable of DNA sequencing. Oxford Nanopore is using protein engineering techniques to adapt the nanopore for the detection of DNA bases.


A key achievement in adapting the nanopore has been the covalent attachment of a cyclodextrin molecule to the inside surface of the nanopore, This acts as a binding site for individual DNA bases and allows accurate measurement of their passage through the nanopore binding site.

Oxford Nanopore is addressing the issue of how DNA is passed through the nanopore by adopting a novel exonuclease sequencing approach. This takes advantage of the ability of exonuclease enzymes to process DNA and cleave individual bases from the end of a DNA strand. By positioning an exonuclease in the correct position on a nanopore, the enzyme can potentially deliver individual DNA bases in sequence into the nanopore for rapid and accurate identification.


Future generations of Oxford Nanopore's sequencing technology
Future generations of the company's technology may feature:

• The ability to sequence intact strands of DNA rather than cleaving individual bases
• a combination of protein nanopore and man-made membrane (instead of lipid bilayers)
• nanopores as holes made directly in man made materials ("solid state" nanopores)

Oxford Nanopore's network of collaborators continue to work on these developments and the company has leading intellectual property in these areas (see press releases: 1, 2)


The DNA sequencing system
The Company's first generation of DNA sequencing system includes the following elements:

Nanopore	Array Chip	Hardware
A protein nanopore, alpha-hemolysin An exonuclease is coupled with the nanopore. This enzyme is responsible for capturing DNA strands, sequentially cleaving individual bases from the strand, and directing the bases into the aperture of the nanopore. An engineered cyclodextrin sensor is covalently attached to the inside surface of the nanopore. This acts as a binding site for DNA bases as they pass through the pore. click here for more information on nanopore chemistry.	image: fluorescent image showing part of Oxford Nanopore's proprietary array chip. Each microwell is an individually recordable electronic channel A lipid bilayer is created over a microwell that contains a pair of electrodes on either side of the bilayer. Adapted alpha-hemolysin nanopores are introduced into the bilayer, creating a single hole. The lipid bilayer has a high electrical resistance and so when an electrical potential is applied across this membrane, a current flows only through the nanopore, carried by the ions in salt solutions that bathe both sides of the bilayer. DNA sample is introduced into the top layer. As the exonuclease directs individual DNA bases, in sequence, through the nanopore, each base transiently binds at the binding site. During the binding event, the current through the nanopore is disturbed, creating a characteristic signal for each type of base. The signal for each base can be easily distinguished. The electrical current trace provides a record of the sequence of bases passing through the nanopore. Click here for more information about our array chip.	To achieve high-throughput sequencing, this system is run in parallel in an array chip. By combining the traces from each well in the array chip, data reassembly can be performed and the genome sequence will be constructed. Simple instrumentation is required to operate the array chip, and record the resulting electrical signals. Direct electrical detection and potential long read lengths promises simpler bioinformatics click here for more information about nanopore informatics