Monthly Archives: September 2012

ScienceDaily (Sep. 21, 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.

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, from Nature Publishing 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 -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.

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Novel approach for single molecule electronic DNA sequencing

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.

(Phys.org)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.

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, 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 -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.

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.

More information: Scientific Reports, 2, 684 DOI:10.1038/srep00684, 2012

Journal reference: Scientific Reports

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Researchers report novel approach for single molecule electronic DNA sequencing

A Middlesex Superior Court judge is allowing two samples of DNA to be used as evidence in the trial of a Framingham man accused of murdering a couple in Hudson in 2010.

Judge Sandra Hamlin denied defense attorney Thomas Fords request to dismiss a sample of Velezs DNA that was found underneath Trisha Bennetts fingernail and a blood spatter found on the jeans Velez wore the night of the murders, said Stephanie Chelf Guyotte, a spokeswoman for the Middlesex District Attorneys office.

During a pre-trial conference earlier this week, Ford argued that a report did not note which portion of Bennetts fingernail the DNA sample was taken from. Ford said DNA can be transmitted to the top of another persons fingernail through casual contact. However, DNA is normally transmitted underneath another persons fingernail if there is sexual or defensive contact.

Ford also expressed concerns that there was no defense expert present at the swabbing and testing of the DNA.

Assistant District Attorney Joseph Gentile said testimony at a previous hearing documented the DNA sample was found underneath Bennetts fingernail.

In the case of the blood spatter on Velezs jeans, Ford said a report did not identify which blood spot was extracted and tested for DNA. The number of spots tested was also not in the report, said Ford.

Citing a report, Gentile said the sample was taken from a defined section near the left thigh Velezs jeans near his thigh.

Jury selection began Friday and will continue on Monday, said Guyotte.

Velez, 29, is charged with first-degree murder in the stabbing deaths of Bennett, 20, and her boyfriend Angel Ortiz, 23. Bennett and Ortiz were found dead inside their Emerson Gardens condominium May 1, 2010.

Authorities arrested Velez nearly three months after he called 911 in the early morning of May 1, 2010, telling police he and two friends had been stabbed. Police found Velez, who was suffering from stab wounds, in the parking lot. Prosecutors say Velezs wounds were self-inflicted.

Continued here:
Judge denies motions to dismiss DNA evidence in Hudson murder case

It's history being made -- the FBI just this month took acceptance of its first-ever "Rapid DNA" equipment for near-instant DNA analysis in the field. But use of this DNA analysis-in-a-box, which can be carried around and connected to the Internet, may be slowed because current law never envisioned such analysis being done for law-enforcement purposes outside an accredited lab.

RELATED: FBI eager to embrace 'Rapid DNA' testing

That realization, brought to light at the Biometric Consortium Conference on Wednesday, cast a shadow on what's a shining moment for the biometrics industry and its partnership with the FBI. The FBI has spent years working to build Rapid DNA equipment according to careful designs for ruggedness, security and usefulness in generating individual DNA profile data that police stations could use to share and match against the FBI's existing DNA Index System (NDIS) database. Such Rapid DNA gear can take in a cotton swab of an individual's saliva or blood in the field and within about 90 minutes, automatically spit out a human DNA profile.

Dr. Thomas Callaghan , senior biometric scientist in the biometric analysis section of the FBI Laboratory, just this month took delivery on the first two working models of Rapid DNA machines, the RapidHit 200 made by integenX, and the ANDE box made by NetBio. "It really is a remarkable achievement," says Callaghan. He and many others in the biometrics field this week at the conference recognized the historic significance of the technology breakthrough presented by the first commercially-viable equipment for Rapid DNA.

The U.S. Army has started evaluation of two ANDE System boxes it got from NetBio, says Jeff Salyards, chief scientist at the U.S. Army Criminal Investigative Laboratory. He reports that the Rapid DNA technology supplied by ANDE appears to work effectively.

Richard Selden, CEO of NetBio, assures that the ANDE System boxes for DNA analysis have undergone military-standard testing for ruggedness. However, Salyards says more testing is needed, and cautioned military buyers, eager to use Rapid DNA equipment in the field, to show patience as more testing is done.

It also could be a while until Rapid DNA can be used for U.S. law enforcement purposes. The National Institute of Standards and Technology (NIST), which is teaming with the FBI to test the NetBio and IntegenX systems, as well as possibly others, for use with law enforcement, expects a full evaluation that includes new processes to be followed to connect to federal databases. Such an evaluation could take upwards of a year.

What's more, the DNA Identification Act of 1994 passed by Congress gave the FBI the authority to establish its DNA index system, but didn't envision that DNA information would be uploaded to the FBI database from a police station using Internet-connected Rapid DNA equipment. The law covers only accredited DNA labs in use today, not the mobile Rapid DNA equipment that can be operated by non-technical personnel anywhere, according to Clark Jaw, an auditor at the FBI Laboratory for the Combined DNA Index System (CODIS). It appears there needs to be a change to the DNA Identification Act to accommodate use of the new technology, he says.

Other obstacles to achieve full-scale use in law enforcement include the need to build out CODIS software to accommodate Rapid DNA and create a quality-assurance process system. That all means Rapid DNA for law-enforcement purposes in the U.S. may take time. But the first Rapid DNA equipment is known to already be in use among secretive intelligence agencies.

"The ultimate goal is to have that technology available for law enforcement use at the police station," Jaw says, pointing out that one day law enforcement officials should be able to carry out real-time DNA-related searches using the Rapid DNA equipment to aid in fast investigation of crime suspects and crime scenes.

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Legal hurdles threaten to slow FBI's 'Rapid DNA' revolution