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DNA sequencing using helicase-modified alpha-hemolysin nanopores

Final Report Summary - HELIPORE (DNA sequencing using helicase-modified alpha-hemolysin nanopores)

Nanopores technology for DNA sequencing and stochastic sensing
The development of cheap and rapid methods for DNA sequencing is impacting many aspects of life, such as: life sciences, medical diagnostics, pharmaceutical and forensics. In 2004 the American NIH set the goal of a $1,000 cost for full human genome sequence, to be achieved by 2015. Nanopore-based DNA sequencing has the potential for ultra-fast and cheap DNA sequencing. Two main approaches exist: (i) Solid-state nanopores fabricated in various robust materials including SiN, glass and polycarbonate, and (ii) biological nanopores, such as α-hemolysin (αHL) or MSPA. Protein nanopores have advantages over solid-state pores, notably the ability to genetically engineer mutations that incorporate functional amino acid residues (e.g. cysteine) at desired positions within the pore lumen. Another advantage of biological nanopores over solid state nanopores is the low cost of manufacture. Moreover, biological nanopores are identical (in terms of their structure) unlike solid state nanopores. In recent years, protein nanopores have been widely used for the development of stochastic sensors for analytes, such as divalent metal ions, biological phosphate compounds, various drugs, proteins, etc. Recently, the αHL nanopore system was shown to sequence long strands of DNA. In 2005 Prof. Hagan Bayley founded a spin-off company – Oxford Nanopores Technologies (ONT) that is focusing on engineering chips for DNA sequencing, based on technology developed in our lab. ONT was able to sequence the whole genome of the λ phage using protein nanopores. In a nanopore sensor device, Fig. 1, a single pore is embedded within a barrier such as a lipid bilayer that separates two compartments. Each compartment contains an Ag/AgCl electrode. The analyte molecule (in our case DNA) is added to the cis side of the nanopore (the cis compartment) and an electric potential is applied at the trans compartment, leading to the translocation of the analyte (DNA) molecule from the cis to the trans side through the nanopore (in the case of DNA, the negatively charged DNA is attracted towards a positive potential applied at the trans chamber). During translocation the DNA molecule partially blocks the ionic current flow. Changes in the residual ionic current are measured and identify the DNA base. In my work I am trying to construct arrays of αHL nanopores on DNA scaffolds for parallel DNA sequencing and sensing devices.