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H I S T O R I C A L P E R S P E C T I V E
Three decades of nanopore sequencing
npg 201 6 Nature America, Inc. All rights reserved.
David Deamer1, Mark Akeson1 & Daniel Branton2
A long-held goal in sequencing has been to use a voltage-biased nanoscale pore in a membrane to measure the passage of a linear, single-stranded (ss) DNA or RNA molecule through that pore. With the development of enzyme-based methods that ratchet polynucleotides through the nanopore, nucleobaseby-nucleobase, measurements of changes in the current through the pore can now be decoded into a DNA sequence using an algorithm. In this Historical Perspective, we describe the key steps in nanopore strand-sequencing, from its earliest conceptualization more than 25 years ago to its recent commercialization and application.
Nanopore sequencing (Box 1 and Fig. 1) has its origins in several laboratories during the 1980s (Fig. 2). In 1989, one of us (D.D.) jotted a seemingly implausible idea in his notebook (Fig. 3), suggesting that it might be possible to sequence a single strand of DNA being drawn through a membranes nanoscopic pore by electrophoresis. Around the same time, George Churchs interest in scaling up DNA sequencing for the Human Genome Project led him to propose that an electronically monitored phage motor embedded in a bilayer might provide sequence information upon processing of double-stranded (ds) DNA. Hagan Bayleys interest in membrane proteins and the structure and assembly of oligomeric transmembrane protein pores, such as a-hemolysin, also led him and his colleagues to ask if pores could serve as biosensors of molecules and cations1.
Whereas Church decided to pursue other high-speed sequencing methods, two of us (D.D. and D.B.) had independently noted publications suggesting that a-hemolysin from Staphylococcus aureus could form transmembrane channels wide enough (>1.2 nm) to accommodate a single strand of DNA2. Furthermore, D.D. had listened to talks by John Kasianowicz at two scientific conferences and knew that he was working with a-hemolysin. In late 1993, D.D. visited Kasianowicz at the US National Institute of Standards and Technology (NIST) to test whether it was possible to electrophorese homopolymers of RNA through an a-hemolysin pore. Kasianowicz had already gained much experience working with a-hemolysin in collaboration with Bayley,
and together...