Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Researchers Report Novel Approach for Single Molecule Electronic DNA Sequencing

24.09.2012
DNA sequencing is the driving force behind key discoveries in medicine and biology. For instance, the complete sequence of an individual’s genome provides important markers and guidelines for medical diagnostics and healthcare.

Up to now, the major roadblock has been the cost and speed of obtaining highly accurate DNA sequences. While numerous advances have been made in the last 10 years, most current high-throughput sequencing instruments depend on optical techniques for the detection of the four building blocks of DNA: A, C, G and T.

To further advance the measurement capability, electronic DNA sequencing of an ensemble of DNA templates has also been developed. Recently, it has been shown that DNA can be threaded through protein nanoscale pores under an applied electric current to produce electronic signals at single molecule level.

However, because the four nucleotides are very similar in their chemical structures, they cannot easily be distinguished using this technique. Thus, the research and development of a single-molecule electronic DNA sequencing platform is the most active area of investigation and has the potential to produce a hand-held DNA sequencer capable of deciphering the genome for personalized medicine and basic biomedical research.

Schematic of single molecule DNA sequencing by a nanopore with phosphate-tagged nucleotides. Each of the four nucleotides will carry a different tag. During SBS, these tags, attached via the terminal-phosphate of the nucleotide, will be released into the nanopore one at a time where they will produce unique current blockade signatures for sequence determination. A large array of such nanopores will lead to high throughput DNA sequencing.

A team of researchers at Columbia University, headed by Dr. Jingyue Ju (the Samuel Ruben-Peter G. Viele Professor of Engineering, Professor of Chemical Engineering and Pharmacology, Director of the Center for Genome Technology and Biomolecular Engineering), with colleagues at the National Institute of Standards and Technology (NIST) led by Dr. John Kasianowicz (Fellow of the American Physical Society), have developed a novel approach to potentially sequence DNA in nanopores electronically at single molecule level with single-base resolution. This work, entitled “PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNA Sequencing by Synthesis” is now available in the open access online journal, Scientific Reports (2, 684 DOI:10.1038/srep00684, 2012),from the Nature Publication group.

The reported nanopore-based sequencing by synthesis (Nano-SBS) strategy can accurately distinguish four DNA bases by detecting 4 different sized tags released from 5’-phosphate-modified nucleotides at the single molecule level for sequence determination. The basic principle of the Nano-SBS strategy is described as follows. As each nucleotide analog is incorporated into the growing DNA strand during the polymerase reaction, its tag is released by phosphodiester bond formation. The tags will enter a nanopore in the order of their release, producing unique ionic current blockade signatures due to their distinct chemical structures, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof-of-principle, the research team attached four different length polymer tags to the terminal phosphate of 2’-deoxyguanosine-5’-tetraphosphate (a modified DNA building block) and demonstrated efficient incorporation of the nucleotide analogs during the polymerase reaction, as well as better than baseline discrimination among the four tags at single molecule level based on their nanopore ionic current blockade signatures. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform.

In previous work, the Center of Genome Technology & Biomolecular Engineering at Columbia University, led by Professor Ju and Dr. Nicholas J. Turro (William P. Schweitzer Professor of Chemistry), developed a four-color DNA sequencing by synthesis (SBS) platform using cleavable fluorescent nucleotide reversible terminators (NRT), which is licensed to Intelligent Bio-Systems, Inc., a QIAGEN company. SBS with cleavable fluorescent NRTs is the dominant approach used in the next generation DNA sequencing systems. Dr. Kasianowicz and his group at NIST pioneered the investigation of nanopores for single molecule analysis. They previously reported that different length polymers, polyethylene glycols (PEGs), could be distinguished by their unique effects on current readings in a á-hemolysin protein nanopores at single molecule level and subsequently developed a theory for the method. Their results provide the proof-of-concept for single molecule mass spectrometry. The combination of the SBS concept with the distinct nanopore-detectable electronic tags to label DNA building blocks led to the development of the single-molecule electronic Nano-SBS approach described the current Scientific Reports article (09/21/2012).

As lead author Dr. Shiv Kumar points out, “The novelty of our approach lies in the design and use of four differently tagged nucleotides, which upon incorporation by DNA polymerase, release four different size tags that are distinguished from each other at the single molecule level when they pass through the nanopore. This approach overcomes any constraints imposed by the small differences among the four nucleotides, a challenge which most nanopore sequencing methods have faced for decades.” Moreover, the technique is quite flexible; with PEG tags as prototypes, other chemical tags can be chosen to provide optimal separation in different nanopore systems.

With further development of this Nano-SBS approach, such as the use of large arrays of protein or solid nanopores, this system has the potential to accurately sequence an entire human genome rapidly and at low cost, thereby enabling it to be used in routine medical diagnoses.

The authors of the Scientific Reports article were Shiv Kumar, Chuanjuan Tao, Minchen Chien, Brittney Hellner, Arvind Balijepalli, Joseph W.F. Robertson, Zengmin Li, James J. Russo, Joseph E. Reiner, John J. Kasianowicz, and Jingyue Ju. The study was supported by a grant from the National Institutes of Health, a National Research Council/NIST/NIH Research Fellowship, and a grant from the NIST Office of Law Enforcement Standards.

Beth Kwon | EurekAlert!
Further information:
http://www.columbia.edu

More articles from Life Sciences:

nachricht The birth of a new protein
20.10.2017 | University of Arizona

nachricht Building New Moss Factories
20.10.2017 | Albert-Ludwigs-Universität Freiburg im Breisgau

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: Neutron star merger directly observed for the first time

University of Maryland researchers contribute to historic detection of gravitational waves and light created by event

On August 17, 2017, at 12:41:04 UTC, scientists made the first direct observation of a merger between two neutron stars--the dense, collapsed cores that remain...

Im Focus: Breaking: the first light from two neutron stars merging

Seven new papers describe the first-ever detection of light from a gravitational wave source. The event, caused by two neutron stars colliding and merging together, was dubbed GW170817 because it sent ripples through space-time that reached Earth on 2017 August 17. Around the world, hundreds of excited astronomers mobilized quickly and were able to observe the event using numerous telescopes, providing a wealth of new data.

Previous detections of gravitational waves have all involved the merger of two black holes, a feat that won the 2017 Nobel Prize in Physics earlier this month....

Im Focus: Smart sensors for efficient processes

Material defects in end products can quickly result in failures in many areas of industry, and have a massive impact on the safe use of their products. This is why, in the field of quality assurance, intelligent, nondestructive sensor systems play a key role. They allow testing components and parts in a rapid and cost-efficient manner without destroying the actual product or changing its surface. Experts from the Fraunhofer IZFP in Saarbrücken will be presenting two exhibits at the Blechexpo in Stuttgart from 7–10 November 2017 that allow fast, reliable, and automated characterization of materials and detection of defects (Hall 5, Booth 5306).

When quality testing uses time-consuming destructive test methods, it can result in enormous costs due to damaging or destroying the products. And given that...

Im Focus: Cold molecules on collision course

Using a new cooling technique MPQ scientists succeed at observing collisions in a dense beam of cold and slow dipolar molecules.

How do chemical reactions proceed at extremely low temperatures? The answer requires the investigation of molecular samples that are cold, dense, and slow at...

Im Focus: Shrinking the proton again!

Scientists from the Max Planck Institute of Quantum Optics, using high precision laser spectroscopy of atomic hydrogen, confirm the surprisingly small value of the proton radius determined from muonic hydrogen.

It was one of the breakthroughs of the year 2010: Laser spectroscopy of muonic hydrogen resulted in a value for the proton charge radius that was significantly...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

ASEAN Member States discuss the future role of renewable energy

17.10.2017 | Event News

World Health Summit 2017: International experts set the course for the future of Global Health

10.10.2017 | Event News

Climate Engineering Conference 2017 Opens in Berlin

10.10.2017 | Event News

 
Latest News

Terahertz spectroscopy goes nano

20.10.2017 | Information Technology

Strange but true: Turning a material upside down can sometimes make it softer

20.10.2017 | Materials Sciences

NRL clarifies valley polarization for electronic and optoelectronic technologies

20.10.2017 | Interdisciplinary Research

VideoLinks
B2B-VideoLinks
More VideoLinks >>>