Reveo’s direct DNA sequencing using tunneling microscopy

Reveo is developing an ambitious technology to stretch out and deposit taut DNA on conductive surfaces for electronic base detection using one or more STM tips and tunneling current measurements. The linearization and deposition of nucleic acid sequences will likely be done using molecular combing. Reveo’s approach requires atomically flat and positively charged substrate surfaces (e.g. self-assembled monolayers on gold substrates or treated graphite substrates). In addition to molecular combing, Reveo has proposed other methods to linearize DNA, including electrophoretic and hydrodynamic stretching and transfer printing[1]. Furthermore, Reveo has proposed to develop STM tips that are knife-edge shaped, where the smallest dimension is nanoscale[2, 3].

Schematic of Reveo's DNA sequencing method from US 2009/0121133

Much like with IBM/Roche’s proposed DNA transistor, Reveo’s competitive advantage is largely based on the potential cost reduction associated with avoiding labels and the possibility of exceptionally long read lengths. In principle, there’s not much difference between Reveo’s technology and that of IBM’s DNA transistor: both stretch and confine DNA to allow for tunneling current measurement of individual bases (albeit in different geometrical arrangements). IBM’s approach will likely be more reproducible and offer a higher degree of control over local DNA segment position. It’s possible that Reveo’s immobilization approach will reduce smaller-scale configurational rearrangement and motion compared to IBM’s approach, but Reveo’s method will likely be undermined by irregularities in DNA deposition. Reveo’s knife-edge tip design is intriguing, as it would avoid cumbersome issues of probe tip and DNA backbone alignment. Interestingly, even with nanoscale knife-edge tips it appears that individual, isolated DNA molecules would be required as a starting point for analysis, as simultaneous analysis of multiple strands would only be feasible if local DNA contour were uniform along an entire chain length (thus permitting the deconvolution of signals from multiple DNA strands given each individual strand would generation a periodic signal).

Outside of its entry into the Archon X Prize for Genomics, little is known about Reveo’s efforts in DNA sequencing. The company was founded in 1991, has spun out a number of companies, and holds over 300 patents across multiple technology and product areas[4]. Furthermore, although Reveo announced a partnership with the University of Washington (Babak Parviz’s lab) in 2006[5], a 2008 Nature Methods article describing Reveo’s technology did not reference the University of Washington[3]. The University of Washington was awarded a $1.5 million grant in 2006 to develop this technology and Reveo has cited this as its own funding [6, 2]. US patent application 2009/121133 described above lists Parviz as the inventor and University of Washington as the assignee; it’s quite possible that Reveo has priority rights to this and related, future University of Washington patents.

Copyright © Bruce A. Schiamberg 2010. All rights reserved.


[1] US 2009/121133


[3] Blow, Nature Methods, vol. 5, p. 267 (2008)




Mobious Biosystems’ single molecule sequencing

Mobious Biosystems was founded in 1999. It is developing instruments to detect single polymerase conformation or mass changes during the sequencing-by-synthesis process, using physical methods not dependent on the use of fluorophores. The start-up does not appear to have developed anything at this time that will significantly impact the third generation sequencing market. It does appear to have made progress on PCR and hybridization array technologies[1]. It is important, however, to note the impressive range of sequencing ideas and corresponding patents generated by founder Daniel Densham and the small company (see below). Conversely, it’s important to recognize that nearly all of Mobious’ technologies either currently are (or initially were) very early-stage and ambitious from a technical standpoint. Its set of proposed technologies span too many areas to be compatible with a start-up’s capabilities and resources. In fact, Mobious’ patent portfolio has the breadth one would expect from the likes of Roche Diagnostics or Life Technologies.

Schematic of SPR technology from US 2008/0014592

The following is a sampling of the core components of Mobious’ proposed sequencing methods and patents: 1) detect conformational changes in a single processing enzyme or a change in the polymerase’s mass (e.g. association with a nucleotide) using SPR, TIRM, or other light-based interrogation methods. In one embodiment, nucleotides are added sequentially, and in another, advanced blocking group chemistry is proposed to allow all nucleotide types to be present in the same reaction[2]; 2) measure polymerase dissociation rate from a target strand, leveraging differences in polymerase dissociation rate which are dependent on the presence or absence of a complementary base[3]; 3) detect conformational changes of an enzyme based on FRET[4]; 4) measure a single polymerase’s dielectric constant in order to detect conformational and/or energy level changes indicative of association with a specific nucleotide type[5]; 5) a variation on US2008/0014592 where a helicase is used to accomplish sequencing[6]; and 6) use of other advanced optical methods to detect enzyme conformational states (recent filing)[7].

At this time, Mobious should narrow its focus to only the most promising of its approaches and applications and open its business model to a variety of commercial partnership structures (if it has not done so already). Although fourth generation sequencing should not be ruled out, proteomics and other life sciences tools are likely a better bet. To access fourth generation sequencing, Mobious needs to be able to demonstrate the essential working components of a sequencing prototype at this time – and there should be clear competitive advantage versus other emerging sequencing technologies. Although many of Mobious’ proposed sequencing methods would be too expensive, time-consuming, or error-prone, there are a few strong approaches in the mix. Finally, it’s worth pointing out the degree of secrecy surrounding the company’s activities:  there is a lack of information available on its current sequencing capabilities; and, although it lists University of Exeter Innovation Centre as its headquarters[1], the Centre’s tenant list does not appear to include Mobious for whatever reason[8].

Copyright © Bruce A. Schiamberg 2010. All rights reserved.



[2] US 2008/0014592

[3] US 7604963

[4] US 2005/0214849

[5] US 2008/0064035

[6] US 2008/0096206

[7] US 2009/0029383


LingVitae’s ‘genetic binary code’

Overview: Norwegian start-up LingVitae is developing a tool to translate biological data of interest into a form that can be more readily detected. It’s using a restriction and ligation enzyme system to cleave two end bases at a time from target DNA fragments of around 4- 40 bases[1], and effectively replace such two base combinations with predetermined, longer sequences of DNA (or DNA bound to labels), which are then concatenated into a new and much longer DNA strand. The process can be thought of as making a genetic binary code, where A, T, G, and C are replaced with, for example, 0-0, 0-1, 1-0, and 1-1 (where 0’s and 1’s correspond to distinct 10-base-long units). The resulting DNA concatemers would be amenable to hybridization with probe oligonucleotides[2]. In addition, a similar overall method could be used to convert protein sequences into nucleic acid sequences for detection[3]. Continue reading

Halcyon Molecular developing electron microscopy-based sequencing

Overview: Start-up Halcyon Molecular is developing a method to sequence nucleic acids using high-atomic-number-labeled bases and electron microscopy. This approach to detection was first proposed by Richard Feynman around 1958. Halycon Molecular is also developing a number of supporting techniques, including use of functionalized needles to stretch and place taut DNA onto substrates for subsequent analysis. The company has around 15 employees and is located in the San Francisco Bay Area.

For additional information on Halcyon Molecular or analysis of related technologies and companies, contact:

Copyright © Bruce A. Schiamberg 2010. All rights reserved.

Ion Torrent developing yet another innovative approach to DNA and RNA analysis

Overview: Ion Torrent’s US 2010/0035252 patent application was published in February 2010. The patent is designed to cover an innovative approach to measurement of base or multiple-base-segment incorporation during sequencing-by-synthesis. The early-stage technology is based on measuring DNA or RNA length in a microfluidic flow cell. Ion Torrent’s proposed technology differs markedly from its core sequencing platform of semiconductor-based ion detection associated with sequencing-by-synthesis – though the technology in the recent filing also obviates or reduces the need for fluorescent labels.

For additional information on Ion Torrent or analysis of related technologies and companies, contact:

Copyright © Bruce A. Schiamberg 2010. All rights reserved.

Brown University’s mass spectrometry for DNA sequencing

Overview: Brown University’s laboratory-stage technology combines nanopores and mass spectrometry for single-molecule DNA sequencing. Tools and diagnostics companies should monitor one form of the proposed technology, which, if successful, would be a major advance for the sequentially-ordered removal of individual bases from DNA strands. Continue reading

IBM’s ‘DNA transistor’

Overview: IBM is developing a nanostructured DNA sequencing device to electronically detect individual bases and rapidly sequence genomes. The technology is innovative yet unproven, and faces major technical hurdles. If experiments are successful, the device will be highly competitive. Its device would be a low-cost alternative to on-market other development-stage technologies at Illumina (ILMN), Life Technologies (LIFE), Pacific Biosciences, Ion Torrent, and other leading companies.

Background: IBM plans to build a DNA sequencing device comprised of nanometer pores with internal surfaces of axially-stratified, alternating nanoscale layers of metal and dielectric material. Individual, in-tact, single-stranded DNA molecules are to be alternatively passed through the pores and held in place for electronic interrogation of individual bases. The approach aims to control DNA’s local motion and configuration at an unprecedented level. IBM has carried out computational and theoretical work, but lacks experimental verification[1]. This is not the first time IBM has ventured into biology. For example, IBM is developing methods to use DNA in the assembly of finer-featured silicon chips[2], has an experimental R&D group focused on microfluidics and biopatterning, and has filed patents covering the manipulation of biological molecules related to the scanning tunneling and related atomic force microscopy technologies that its researchers famously discovered[3].

Analysis: IBM’s cost savings would result from fewer reagents, no need for labels and optical instrumentation, and long read lengths. In comparison to other nanopore-based sequencing technologies (e.g. Oxford Nanopore Technologies/Illumina and NABsys), IBM’s device would be more stable and less expensive. IBM faces two critical technical challenges. Its device must: 1) distinguish the signal of a single base from the signals of nearby surrounding bases in the DNA chain; 2) manipulate and control DNA to slow down translocation and to enable optimal base orientation and/or sufficient sampling of a base throughout its range of configurations.

Regarding the challenge of base measurement, IBM will almost necessarily take a different approach to measurement than Oxford Nanopore, likely involving direct measurement of tunneling currents or capacitance. This approach could obviate limitations of spatial resolution and sensitivity associated with the Oxford Nanopore approach[4], and recent simulations have shown that a host of complicating phenomena such as the effect of ionic motion in a nanopore may not obscure the desired signal of an individual base[5]. Oxford Nanopore does not need to achieve nearly the same degree of measurement sensitivity as IBM, as the current Oxford approach involves interrogation of cleaved bases, and not in-tact DNA strands. NABsys is also taking a very different approach with lower measurement sensitivity requirements. A search of the patent literature reveals the first IBM patent covering its DNA transistor technology[6], and indeed IBM is seeking coverage with its technology of tunneling current and capacitance measurements.

Schematic of nanopore (from IBM's US 2008/0187915)

Regarding the challenge of precise DNA translocation, the axially-stratified layers of IBM’s proposed nanopores represent an entirely new approach to the problem. In its initial patent, IBM presents an innovative approach to manipulate nucleic acids, including voltage application to draw charged polymers into nanopores (or advance segments within pores), in combination with electrostatic potential wells that interdigitate with individual bases for trapping and analysis. The electrostatic wells could be used in combination with other electrostatic wells in the same pore[6]. IBM researchers estimate that it is possible to achieve a frequency of around 500 MHz with such a device, which would translate into a theoretical read rate of 0.5 billion bases per second[7]. Although there are polymer structure limitations which make this exceptionally high rate unlikely, it is clear that voltage cycling times will not be a limiting factor. There are several approaches IBM may take in upcoming experiments. It will need to focus on synchronizing DNA movement by voltage application with base-level movement using potential wells, while ensuring that DNA-wall interactions (possibly affected in a sequence-dependent manner), the time-dependent quantity of DNA on either side of a nanopore during sequencing (and hence changing drag force), and inconsistencies in test fluid composition do not reduce read fidelity below acceptable levels. Finally, fabrication of such a nanostructured material is non-trivial and represents a challenge to IBM, as well as serious barrier to other companies and researchers in the field. IBM researchers indicate several possible fabrication methods, including electron and ion beam techniques[7].

For additional information on IBM’s technology or analysis of related technologies and companies, contact:

Copyright © Bruce A. Schiamberg 2010. All rights reserved.


[3] US 7211789
[4] Branton et. al., Nature Biotechnology, vol. 26, no. 10, p. 1146 (2008)
[5] Krems et. al., Biophysical Journal, vol. 97, no. 7, p.1990 (2009)
[6] US 2008/0187915
[7] Polonsky et. al., IBM Research Report: DNA Transistor, RC24242 (W0704-094), April 2007


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