Nanopore sequencing is a currently under development method for determining the order in which nucleotides occur on a strand of DNA.

A nanopore is simply a small hole, of the order of 1 nanometer in diameter. Certain transmembrane cellular proteins act as nanopores, and nanopores have also been made by etching a somewhat larger hole (several tens of nanometers) in a piece of silicon, and then gradually filling it in using electron beam methods which results in a much smaller diameter hole: the nanopore.

The theory behind nanopore sequencing has to do with what occurs when the nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it: under these conditions a slight electrical current due to conduction of ions through the nanopore can be observed, and the amount of current which flows is exceedingly sensitive to the size of the nanopore. When DNA is present in the solution, electrophoresis means that the DNA molecules will start to migrate towards the nanopore, and eventually pass through it. However, the scale of the nanopore ensures that the DNA is forced through the hole as a long string, one base at a time, rather like thread through the eye of a needle. As it does so, each nucleotide on the DNA molecule obstructs the nanopore to a different, characteristic degree. The amount of current which can pass through the nanopore at any given moment therefore varies depending on whether the nanopore is blocked by an A, a C, a G or a T. The change in the current passing through the nanopore as the DNA molecule passes through the nanopore represents a direct reading of the DNA sequence.

The potential is that a single molecule of DNA can be sequenced from a single pass through a nanopore, and without the need for an intervening PCR amplification step. At this time however, nanopore sequencing is a promising theoretical method, with only limited lab bench results.