Nanopore sequencing
Encyclopedia
Nanopore sequencing is a method under development since 1995 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 internal diameter. Certain porous 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 ion-beam sculpting
methods which results in a much smaller diameter hole: the nanopore. graphene
is also being explored as a synthetic substrate for solid-state nanopores.
The theory behind nanopore sequencing is that when a nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it, an electric current
due to conduction of ions through the nanopore can be observed. The amount of current is very sensitive to the size and shape of the nanopore. If single nucleotides (bases), strands of DNA or other molecules pass through or near the nanopore, this can create a characteristic change in the magnitude of the current through the nanopore.
DNA could be passed through the nanopore for various reasons. For example, electrophoresis
might attract the DNA towards the nanopore, and it might eventually pass through it. Or, enzymes attached to the nanopore might guide DNA towards the nanopore. The scale of the nanopore means that the DNA may be 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 may obstruct 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 through the nanopore as the DNA molecule passes through the nanopore represents a direct reading of the DNA sequence. Alternatively, a nanopore might be used to identify individual DNA bases as they pass through the nanopore in the correct order - this approach has been shown by Oxford Nanopore Technologies and Professor Hagan Bayley
.
The potential is that a single molecule of DNA can be sequenced directly using a nanopore, without the need for an intervening PCR amplification step or a chemical labelling step or the need for optical instrumentation to identify the chemical label. As of July 2010, information available to the public indicates that nanopore sequencing is still in the development stage, with some laboratory-based data to back up the different components of the sequencing method, but not yet commercially available, parallelized, routineized, nor cost-effective enough yet to compete with out "next generation sequencing" methods. Nanopore-based DNA analysis techniques are being industrially developed by Oxford Nanopore Technologies (developing direct exonuclease sequencing and strand sequencing using protein nanopores, and solid-state sequencing through internal R&D and collaborations with academic institutions), NabSys (using a library of DNA probes and using nanopores to detect where these probes have hybridized to single stranded DNA) and NobleGen(using nanopores in combination with fluorescent labels). IBM has noted research projects on computer simulations of translocation of a DNA strand through a solid-state nanopore, but not projects on identifying the DNA bases on that strand.
One challenge for the 'strand sequencing' method is in refining the method to improve its resolution to be able to detect single bases. In the early papers methods, a nucleotide needed to be repeated in a sequence about 100 times successively in order to produce a measurable characteristic change. This low resolution is because the DNA strand moves rapidly at the rate of 1 to 5μs per base through the nanopore. This makes recording difficult and prone to background noise, failing in obtaining single-nucleotide resolution. The problem is being tackled by either improving the recording technology or by controlling the speed of DNA strand by various protein engineering strategies. More recently effects of single bases due to secondary structure or released mononucleotides have been shown. Professor Hagan Bayley, founder of Oxford Nanopore, recently proposed that creating two recognition sites within an alpha hemolysin pore may confer advantages in base recognition.
One challenge for the 'exonuclease approach', where a processive enzyme feeds individual bases, in the correct order, into the nanopore, is to integrate the exonuclease and the nanopore detection systems. In particular, the problem is that when an exonuclease hydrolyzes the phosphodieseter bonds between nucleotides in DNA, the subsequentially released nucleotide is not necessarily guaranteed to directly move in to, say, a nearby alpha-hemolysin nanopore
. One idea is to attach the exonuclease to the nanopore, perhaps through biotinylation
to the beta barrel
hemolsyin. The central pore of the protein may be lined with charged residues arranged so that the positive and negative charges appear on opposite sides of the pore. However, this mechanism is primarily discriminatory and does not constitute a mechanism to guide nucleotides down some particular path.
The company Oxford Nanopore Technologies in 2008 licensed technology from Harvard, UCSC and other universities and is developing protein and solid state nanopore technology with the aim of sequencing DNA and identifying biomarkers, drugs of abuse and a range of other molecules.
Sequenom
licensed nanopore technology from Harvard in 2007 using an approach that combines nanopores and fluorescent labels. This technology was subsequently licensed to Noblegen.
NABsys was spun out of Brown University and is researching nanopores as a method of identifying areas of single stranded DNA that have been hybridized with specific DNA probes.
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
.
A nanopore
Nanopore
A nanopore is a small hole. It may, for example, be created by a pore-forming protein or as a hole in synthetic materials such as silicon or graphene....
is simply a small hole, of the order of 1 nanometer in internal diameter. Certain porous 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 ion-beam sculpting
Ion-beam sculpting
Ion-Beam scultping is a term used to describe a two-step process to make solid-state nanopores. The term itself was coined by Golovchenko and co-workers at Harvard in the paper "Ion-beam sculpting at nanometer length scales." The term refers to the fact that solid-state nanopores are formed by...
methods which results in a much smaller diameter hole: the nanopore. graphene
Graphene
Graphene is an allotrope of carbon, whose structure is one-atom-thick planar sheets of sp2-bonded carbon atoms that are densely packed in a honeycomb crystal lattice. The term graphene was coined as a combination of graphite and the suffix -ene by Hanns-Peter Boehm, who described single-layer...
is also being explored as a synthetic substrate for solid-state nanopores.
The theory behind nanopore sequencing is that when a nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it, an electric current
Electric current
Electric current is a flow of electric charge through a medium.This charge is typically carried by moving electrons in a conductor such as wire...
due to conduction of ions through the nanopore can be observed. The amount of current is very sensitive to the size and shape of the nanopore. If single nucleotides (bases), strands of DNA or other molecules pass through or near the nanopore, this can create a characteristic change in the magnitude of the current through the nanopore.
DNA could be passed through the nanopore for various reasons. For example, electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
might attract the DNA towards the nanopore, and it might eventually pass through it. Or, enzymes attached to the nanopore might guide DNA towards the nanopore. The scale of the nanopore means that the DNA may be 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
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
on the DNA molecule may obstruct 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 through the nanopore as the DNA molecule passes through the nanopore represents a direct reading of the DNA sequence. Alternatively, a nanopore might be used to identify individual DNA bases as they pass through the nanopore in the correct order - this approach has been shown by Oxford Nanopore Technologies and Professor Hagan Bayley
Hagan Bayley
John Hagan Pryce Bayley FRS is a British Professor of Chemical Biology at the University of Oxford. Bayley's research is largely based on the study and engineering of transmembrane pore-forming proteins, as well as interests in chemical signal transduction and biomolecular materials. He is the...
.
The potential is that a single molecule of DNA can be sequenced directly using a nanopore, without the need for an intervening PCR amplification step or a chemical labelling step or the need for optical instrumentation to identify the chemical label. As of July 2010, information available to the public indicates that nanopore sequencing is still in the development stage, with some laboratory-based data to back up the different components of the sequencing method, but not yet commercially available, parallelized, routineized, nor cost-effective enough yet to compete with out "next generation sequencing" methods. Nanopore-based DNA analysis techniques are being industrially developed by Oxford Nanopore Technologies (developing direct exonuclease sequencing and strand sequencing using protein nanopores, and solid-state sequencing through internal R&D and collaborations with academic institutions), NabSys (using a library of DNA probes and using nanopores to detect where these probes have hybridized to single stranded DNA) and NobleGen(using nanopores in combination with fluorescent labels). IBM has noted research projects on computer simulations of translocation of a DNA strand through a solid-state nanopore, but not projects on identifying the DNA bases on that strand.
One challenge for the 'strand sequencing' method is in refining the method to improve its resolution to be able to detect single bases. In the early papers methods, a nucleotide needed to be repeated in a sequence about 100 times successively in order to produce a measurable characteristic change. This low resolution is because the DNA strand moves rapidly at the rate of 1 to 5μs per base through the nanopore. This makes recording difficult and prone to background noise, failing in obtaining single-nucleotide resolution. The problem is being tackled by either improving the recording technology or by controlling the speed of DNA strand by various protein engineering strategies. More recently effects of single bases due to secondary structure or released mononucleotides have been shown. Professor Hagan Bayley, founder of Oxford Nanopore, recently proposed that creating two recognition sites within an alpha hemolysin pore may confer advantages in base recognition.
One challenge for the 'exonuclease approach', where a processive enzyme feeds individual bases, in the correct order, into the nanopore, is to integrate the exonuclease and the nanopore detection systems. In particular, the problem is that when an exonuclease hydrolyzes the phosphodieseter bonds between nucleotides in DNA, the subsequentially released nucleotide is not necessarily guaranteed to directly move in to, say, a nearby alpha-hemolysin nanopore
Hemolysin
Hemolysins are exotoxins produced by bacteria that cause lysis of red blood cells in vitro. Visualization of hemolysis of red blood cells in agar plates facilitates the categorization of some pathogenic bacteria such as Streptococcus and Staphylococcus...
. One idea is to attach the exonuclease to the nanopore, perhaps through biotinylation
Biotinylation
In biochemistry, biotinylation is the process of covalently attaching biotin to a protein, nucleic acid or other molecule. Biotinylation is rapid, specific and is unlikely to perturb the natural function of the molecule due to the small size of biotin...
to the beta barrel
Beta barrel
A beta barrel is a large beta-sheet that twists and coils to form a closed structure in which the first strand is hydrogen bonded to the last.Beta-strands in beta-barrels are typically arranged in an antiparallel fashion...
hemolsyin. The central pore of the protein may be lined with charged residues arranged so that the positive and negative charges appear on opposite sides of the pore. However, this mechanism is primarily discriminatory and does not constitute a mechanism to guide nucleotides down some particular path.
Commercialization
Agilent Laboratories was the first to license and develop nanopores but does not have any current disclosed research in the area.The company Oxford Nanopore Technologies in 2008 licensed technology from Harvard, UCSC and other universities and is developing protein and solid state nanopore technology with the aim of sequencing DNA and identifying biomarkers, drugs of abuse and a range of other molecules.
Sequenom
Sequenom
Sequenom is a manufacturer of DNA massarrays, based in San Diego, California, United States. The MassARRAY platform is used for SNP genotyping, methylation detection and quantitative gene expression analysis. Sequenom also manufactures clinical tests, such as SEQureDx, a noninvasive prenatal test...
licensed nanopore technology from Harvard in 2007 using an approach that combines nanopores and fluorescent labels. This technology was subsequently licensed to Noblegen.
NABsys was spun out of Brown University and is researching nanopores as a method of identifying areas of single stranded DNA that have been hybridized with specific DNA probes.
Reviews
- Zwolak M, Di Ventra M. Colloquium: Physical approaches to DNA sequencing and detection. Reviews of Modern Physics 80, 141 (2008)
- Astier Y, Braha O, Bayley H: Towards single molecule DNA sequencing. J. AM. CHEM. SOC. 2006, 128, 1705-1710 9 1705
- Fologea D el al. Detecting single stranded DNA with a solid state nanopore. Nano Lett. 2005 Oct;5(10):1905-9. PMID 16218707
- Deamer DW, Akeson M. Nanopores and nucleic acids: prospects for ultrarapid sequencing. Trends Biotechnol. 2000 Apr;18(4):147-51. PMID 10740260
- Church, George M. Genomes for all. Scientific American. 2006 Jan;294(1):52. PMID 16468433
- Xu M. S., Fujita D., Hanagata N. "Perspectives and challenges of emerging single-molecule DNA sequencing technologies". Small 2009, 5 (23), 2638-49.